Ion exchange chromatography of GLP-1, analogs and derivatives thereof

ABSTRACT

The present invention relates to an ion exchange chromatography process for purifying GLP-1 or an analog or a derivative thereof from a mixture containing said GLP-1 and related impurities, and to an industrial method including such ion exchange chromatography process.

FIELD OF THE INVENTION

[0001] The present invention relates to an ion exchange chromatographyprocess for purifying GLP-1 or an analog or a derivative thereof from amixture containing said GLP-1 and related impurities, and to anindustrial method including such ion exchange chromatography process.

BACKGROUND

[0002] For the purification and analysis of proteins and peptides,chromatography is a well-known and widely used method. A number ofdifferent chromatographic principles are applied, among these ionexchange chromatography (IEC). The IEC principle includes two differentapproaches: anion exchange and cation exchange according to the chargeof the ligands on the ion exchange resin. A conventional IECpurification process usually consists of one or more: equilibrationsections, application or loading sections, wash sections, elutionsections, and regeneration sections (cf. Remington's PharmaceuticalSciences, Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, orRemington: The Science and Practice of Pharamacy, 19th Edition (1995)).

[0003] The main principle of elution in IEC in industrial purificationprocesses is salt component gradients in an aqueous buffer solution atconstant pH, either as step or linear gradients (cf. S. Bjørn and L.Thim, Activation of Coagulation Factor VII to VIIa, Res. Discl. No. 269,564-565, 1986). Isocratic elution is possible, but seldom used. Organicsolvents or modifiers have occasionally been added to the solutions tokeep the protein or peptide on the desired form or just in solution (cf.K. H. Jørgensen, Process for Purifying Insulin, U.S. Pat. No. 3,907,676,Sep. 23, 1975; and J. Brange, O. Hallund and E. Sørensen, ChemicalStability of Insulin 5. Isolation, Characterisation and Identificationof Insulin Transformation Products, Acta Pharm. Nord. 4(4), 223-232,1992).

[0004] Glucagon-Like Peptide-1 (GLP-1) (cf. Schmidt et al. inDiabetologia 28 704-707, 1985) and analogues as well as derivativesthereof may be used in the treatment of diabetes, as disclosed in WO98/08871. A GLP-1 peptide and related analogues are easily dissolved inaqueous solvents and kept in a monomized form. Traditional IECpurification of GLP-1 with salt gradients in aqueous solvents may,however, be problematic due to the lack of selectivity between a GLP-1target moiety and related impurities.

[0005] WO 87/06941 (The General Hospital Corporation) disclose peptidefragments which comprises GLP-1 (7-37) and functional derivativesthereof and to its use as an insulinotropic agent.

[0006] WO 90/11296 (The General Hospital Corporation) disclose peptidefragments which comprise GLP-1 (7-36) and functional derivatives thereofand have an insulinotropic activity which exceeds the insulinotropicactivity of GLP-1(1-36) or GLP-1(1-37) and to their use asinsulinotropic agents.

[0007] WO 91/11457 (Buckley et al.) discloses analogues of the activeGLP-1 peptides 7-34, 7-35, 7-36, and 7-37.

[0008] WO 98/08871 discloses GLP-1 derivatives in which a lipophilicsubstituent is attached to at least one amino acid residue. Thelipophilic substituents are in particular long-chain groups containinge.g. 12-24 carbon atoms.

[0009] WO 98/08872 discloses GLP-2 derivatives in which a lipophilicsubstituent is attached to at least one amino acid residue. Thelipophilic substituents are in particular long-chain groups containinge.g. 12-24 carbon atoms.

[0010] WO 96/32414 discloses GLP-2 analogues.

[0011] EP 0699686-A2 (Eli Lilly & Co.) discloses certain N-terminaltruncated fragments of GLP-1 that are reported to be biologicallyactive.

[0012] EP 0708179-A2 (Eli Lilly & Co.) discloses GLP-1 analogues andderivatives that include an N-terminal imidazole group and optionally anunbranched C₆-C₁₀ acyl group in attached to the lysine residue inposition 34.

DESCRIPTION OF THE INVENTION

[0013] In contrast to the above described IEC techniques forpurification of any protein or peptide, consisting of one or moreequilibration steps, application or loading steps, wash steps, elutionsteps, and regeneration steps, the instant invention relates to the useof an organic modifier for the purification of a GLP-1 peptide and allrelated analogues etc. by IEC. By addition of an organic modifierespecially to the elution section of the IEC purification step, anincrease in selectivity and efficiency is obtained compared to the samerun with aqueous buffers, both for anion and cation exchangechromatography. The equilibration solution and the sample forapplication may or may not contain the organic modifier. The use of anorganic modifier has the additional advantage that no salt or very lowconcentrations of salt is needed for elution, especially for cationexchange chromatography compared to an aqueous chromatographic system.

[0014] In a broad aspect the present invention relates to a cationexchange chromatography process for purifying a peptide from a mixturecomprising said peptide and related impurities, comprising the step of:

[0015] separating said peptide and said related impurities of saidmixture by elution in a solution comprising an organic modifier, water,optionally a salt component, and optionally a buffer, with a linear orstep gradient or isocratically in salt component and/or with a linear orstep pH-gradient or at a constant pH-value, wherein the pH-gradient orpH-value should be in the range where said peptide has a positive localor overall net charge different from the local or overall positive netcharge of said related impurities so as to remove said relatedimpurities.

[0016] In another broad aspect the present invention relates to a cationexchange chromatography process for purifying a peptide from a mixturecomprising said peptide and related impurities, comprising the step of:

[0017] separating said peptide and said related impurities of saidmixture by elution in a solution consisting essentially of an organicmodifier, water, optionally a salt component, and optionally a buffer,with a linear or step gradient or isocratically in salt component and/orwith a linear or step pH-gradient or at a constant pH-value, wherein thepH-gradient or pH-value should be in the range where said peptide has apositive local or overall net charge different from the local or overallpositive net charge of said related impurities so as to remove saidrelated impurities.

[0018] In another broad aspect the present invention relates to an anionexchange chromatography process for purifying a peptide from a mixturecomprising said peptide and related impurities, comprising the step of:

[0019] separating said peptide and said related impurities of saidmixture by elution in a solution comprising an organic modifier, water,optionally a salt component, and optionally a buffer, with a linear orstep gradient or isocratically in salt component and/or with a linear orstep pH-gradient or at a constant pH-value, wherein the pH-gradient orpH-value should be in the range where said peptide has a negative localor overall net charge different from the local or overall negative netcharge of said related impurities so as to remove said relatedimpurities.

[0020] In another broad aspect the present invention relates to an anionexchange chromatography process for purifying a peptide from a mixturecomprising said peptide and related impurities, comprising the step of:

[0021] separating said peptide and said related impurities of saidmixture by elution in a solution consisting essentially of an organicmodifier, water, optionally a salt component, and optionally a buffer,with a linear or step gradient or isocratically in salt component and/orwith a linear or step pH-gradient or at a constant pH-value, wherein thepH-gradient or pH-value should be in the range where said peptide has anegative local or overall net charge different from the local or overallnegative net charge of said related impurities so as to remove saidrelated impurities.

[0022] In an embodiment of the present invention the peptide to bepurified is selected from polypeptides, oligopeptides, proteins,receptors, vira, as well as homologues, analogues and derivativesthereof, preferably glucagon, hGH, insulin, aprotinin, FactorVII, TPA,FactorVIIa (NovoSeven®, available from Novo Nordisk A/S, Bagsvaerd,Denmark), FactorVIIaI, FFR-FactorVIIa, heparinase, ACTH, Heparin BindingProtein, corticotropin-releasing factor, angiotensin, calcitonin,insulin, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2(GLP-2), insulin-like growth factor-1, insulin-like growth factor-2,fibroblast growth factors, gastric inhibitory peptide, growthhormone-releasing factor, pituitary adenylate cyclase activatingpeptide, secretin, enterogastrin, somatostatin, somatotropin,somatomedin, parathyroid hormone, thrombopoietin, erythropoietin,hypothalamic releasing factors, prolactin, thyroid stimulating hormones,endorphins, enkephalins, vasopressin, oxytocin, opiods, DPP IV,interleukins, immunoglobulins, complement inhibitors, serpin proteaseinhibitors, cytokines, cytokine receptors, PDGF, tumor necrosis factors,tumor necrosis factors receptors, growth factors and analogues as wellas derivatives thereof, more preferably glucagon, hGH, insulin,aprotinin, FactorVII, FactorVIIa, FFR-FactorVIIa, heparinase,glucagon-like peptide-1, glucagon-like peptide-2 and analogues as wellas derivatives thereof, such as Arg³⁴GLP-1₍₇₋₃₇₎, human insulin, andB28IsoAsp insulin. Each of these peptides constitutes an alternativeembodiment of the present invention.

[0023] Accordingly, an aspect of the present invention relates to acation exchange chromatography process for purifying a GLP-1 peptidefrom a mixture comprising said peptide and related impurities,comprising the step of:

[0024] separating said GLP-1 peptide and said related impurities of saidmixture by elution in a solution comprising an organic modifier, water,optionally a salt component, and optionally a buffer, with a linear orstep gradient or isocratically in salt component and/or with a linear orstep pH-gradient or at a constant pH-value, wherein the pH-gradient orpH-value should be in the range where said peptide has a positive localor overall net charge different from the local or overall positive netcharge of said related impurities so as to remove said relatedimpurities.

[0025] Another aspect of the present invention relates to a cationexchange chromatography process for purifying a GLP-1 peptide from amixture comprising said peptide and related impurities, comprising thestep of:

[0026] separating said GLP-1 peptide and said related impurities of saidmixture by elution in a solution consisting essentially of an organicmodifier, water, optionally a salt component, and optionally a buffer,with a linear or step gradient or isocratically in salt component and/orwith a linear or step pH-gradient or at a constant pH-value, wherein thepH-gradient or pH-value should be in the range where said peptide has apositive local or overall net charge different from the local or overallpositive net charge of said related impurities so as to remove saidrelated impurities.

[0027] Another aspect of the present invention relates to an anionexchange chromatography process for purifying a GLP-1 peptide from amixture comprising said peptide and related impurities, comprising thestep of:

[0028] separating said GLP-1 peptide and said related impurities of saidmixture by elution in a solution comprising an organic modifier, water,optionally a salt component, and optionally a buffer, with a linear orstep gradient or isocratically in salt component and/or with a linear orstep pH-gradient or at a constant pH-value, wherein the pH-gradient orpH-value should be in the range where said peptide has a negative localor overall net charge different from the local or overall negative netcharge of said related impurities so as to remove said relatedimpurities.

[0029] Another aspect of the present invention relates to an anionexchange chromatography process for purifying a GLP-1 peptide from amixture comprising said peptide and related impurities, comprising thestep of:

[0030] separating said GLP-1 peptide and said related impurities of saidmixture by elution in a solution consisting essentially of an organicmodifier, water, optionally a salt component, and optionally a buffer,with a linear or step gradient or isocratically in salt component and/orwith a linear or step pH-gradient or at a constant pH-value, wherein thepH-gradient or pH-value should be in the range where said peptide has anegative local or overall net charge different from the local or overallnegative net charge of said related impurities so as to remove saidrelated impurities.

[0031] The elution in above aspects may also be possible by changing thecontent of organic modifier in the elution section, which is consideredan embodiment of the present invention.

[0032] The linear or step gradient in the salt component would be from alower to a higher concentration in both IEC modes.

[0033] In the above aspects of the present process the elution couldalso be considered a washing step of related impurities.

[0034] In one embodiment of the present invention the ratio of organicmodifier to water, on a weight percent basis, is from 1:99 to 99:1, suchas 1:99 to 80:20, 20:80 to 80:20, 30:70 to 70:30, 35:50 to 50:35, or40:50 to 50:40. Each of these ratios constitutes an alternativeembodiment of the present invention.

[0035] In another embodiment of the present invention the organicmodifier is selected from C₁₋₆-alkanol, C₁₋₆-alkenol or C₁₋₆-alkynol,urea, guanidine, or C₁₋₆-alkanoic acid, such as acetic acid,C₂₋₆-glycol, C₃₋₇-polyalcohol including sugars, preferably C₁₋₆-alkanoland C₂₋₆-glycol, more preferably methanol, ethanol, propanols andbutanols and hexyl glycols, most preferably ethanol and 2-propanol. Eachof these organic modifiers constitutes an alternative embodiment of thepresent invention.

[0036] In a further embodiment of the present invention the step orlinear pH gradient for the anion exchange chromatography process goesfrom a higher to a lower pH.

[0037] In a further embodiment of the present invention the step orlinear pH gradient for the cation exchange chromatography process goesfrom a lower to a higher pH.

[0038] In a further embodiment of the present invention the saltcomponent is selected from any organic or inorganic salt and mixturesthereof, preferably NaCl, KCl, NH₄Cl, CaCl₂, sodium acetate, potassiumacetate, ammonium acetate, sodium citrate, potassium citrate, ammoniumcitrate, sodium sulphate, potassium sulphate, ammonium sulphate, calciumacetate, or mixtures thereof, most preferred sodium acetate, potassiumacetate, ammonium acetate, NaCl, NH₄Cl, KCl. Each of these saltcomponents constitutes an alternative embodiment of the presentinvention.

[0039] In a further embodiment of the present invention the gradient insalt component is a step gradient in salt component.

[0040] In a further embodiment of the present invention the saltcomponent is present in a step concentration selected from the range of0.1 mmol/kg to 3000 mmol/kg, preferably 1 mmol/kg to 1000 mmol/kg, morepreferably 5 mmol/kg to 500, most preferably 20 mmol/kg to 300 mmol/kg.Each of these ranges constitutes an alternative embodiment of thepresent invention.

[0041] In a further embodiment of the present invention the gradient insalt component is a linear gradient in salt component.

[0042] In a further embodiment of the present invention the saltcomponent is present in a linear concentration selected from 0.1 mmol/kgto 3000 mmol/kg, preferably 1 mmol/kg to 1000 mmol/kg, more preferably 5mmol/kg to 500, most preferably 20 mmol/kg to 300 mmol/kg. Each of theselinear concentrations constitutes an alternative embodiment of thepresent invention.

[0043] In a further embodiment of the present invention no saltcomponent is present.

[0044] In a further embodiment of the present invention the buffer isselected from citrate buffers, phosphate buffers, tris buffers, boratebuffers, lactate buffers, glycyl glycin buffers, arginine buffers,carbonate buffers, acetate buffers, glutamate buffers, ammonium buffers,glycin buffers, alkylamine buffers, aminoethyl alcohol buffers,ethylenediamine buffers, triethanol amine, imidazole buffers, pyridinebuffers and barbiturate buffers and mixtures thereof, preferably citricacid, sodium citrate, potassium citrate, sodium phosphate, potassiumphosphate, phosphorous acid, glutamic acid, sodium glutamate, potassiumglutamate, glycin, sodium carbonate, tris-hydroxymethyl amino methaneand boric acid and mixtures thereof. Each of these buffers constitutesan alternative embodiment of the present invention.

[0045] In a further embodiment of the present invention the buffer ispresent in a concentration selected from the range of 0.1 mmol/kg to 500mmol/kg, preferably 1 mmol/kg to 200 mmol/kg, more preferably 5 mmol/kgto 100 mmol/kg, most preferably 10 mmol/kg to 50 mmol/kg. Each of theseranges constitutes an alternative embodiment of the present invention.

[0046] In a further embodiment of the present invention no buffer ispresent.

[0047] In a further embodiment of the present invention the GLP-1peptide to be purified is selected from GLP-1 (7-37), GLP-1 (7-36) amideas well as analogues and derivatives thereof, in particular but notlimited to human glucagon-like peptide-1, Arg²⁶-GLP-1(7-37);Arg³⁴-GLP-1(7-37); Lys³⁶-GLP-1(7-37); Arg^(26,34)Lys³⁶-GLP-1(7-37);Arg^(26,34)Lys³⁸-GLP-1(7-38); Arg^(26,34)Lys³⁹-GLP-1(7-39);Arg^(26,34)Lys⁴⁰-GLP-1(7-40); Arg²⁶Lys³⁶-GLP-1(7-37);Arg³⁴Lys³⁶-GLP-1(7-37); Arg²⁶Lys³⁹-GLP-1(7-39); Arg³⁴Lys⁴⁰-GLP-1(7-40);Arg^(26,34)Lys^(36,39)-GLP-1(7-39); Arg^(26,34)Lys^(36,40)-GLP-1(7-40);Gly⁸Arg26-GLP-1(7-37); Gly⁸Lys³⁴-GLP-1(7-37);Gly⁸Arg³⁴Lys³⁶-GLP-1(7-37); Gly⁸Arg^(26,34)Lys³⁶-GLP-1(7-39);Gly⁸Arg^(26,34)Lys³⁹-GLP-1(7-39); Gly⁸Arg^(26,34)Lys⁴⁰-GLP-1(7-40);Gly⁸Arg²⁶Lys³⁶GLP-1(7-37); Gly⁸Arg³⁴Lys³⁶-GLP-1(7-37);Gly⁸Arg²⁶Lys³⁹-GLP-1(7-39); Gly⁸Arg³⁴Lys⁴⁰-GLP-1(7-40);Gly⁸Arg^(26,34)Lys^(36,39)-GLP-1-(7-39);Gly⁸Arg^(26,34)Lys^(36,40)-GLP-1(7-40); Arg^(26,34)Lys³⁸GLP-1(7-38);Arg^(26,34)Lys³⁹GLP-1(7-39); Arg^(26,34)Lys⁴⁰GLP-1(7-40);Arg^(26,34)Lys⁴¹GLP-1(7-41); Arg^(26,34)Lys⁴²GLP-1(7-42);Arg^(26,34)Lys⁴³GLP-1(7-43); Arg^(26,34)Lys⁴⁴GLP-1(7-44);Arg^(26,34)Lys⁴⁵GLP-1(7-45); Arg^(26,34)Lys³⁸GLP-1(1-38);Arg^(26,34)Lys³⁹GLP-1(1-39); Arg^(26,34)Lys⁴⁰GLP-1(1-40);Arg^(26,34)Lys⁴¹GLP-1(1-41); Arg^(26,34)Lys⁴²GLP-1(1-42);Arg^(26,34)Lys⁴³GLP-1(1-43); Arg^(26,34)Lys⁴⁴GLP-1(1-44);Arg^(26,34)Lys⁴⁵GLP-1(1-45); Arg^(26,34)Lys³⁸GLP-1(2-38);Arg^(26,34)Lys³⁹GLP-1(2-39); Arg^(26,34)Lys⁴⁰GLP-1(2-40);Arg^(26,34)Lys⁴¹GLP-1(2-41); Arg^(26,34)Lys⁴²GLP-1(2-42);Arg^(26,34)Lys⁴³GLP-1(2-43); Arg^(26,34)Lys⁴⁴GLP-1(2-44);Arg^(26,34)Lys⁴⁵GLP-1(2-45); Arg^(26,34)Lys³⁸GLP-1(3-38);Arg^(26,34)Lys³⁹GLP-1(3-39); Arg^(26,34)Lys⁴⁰GLP-1(3-40);Arg^(26,34)Lys⁴¹GLP-1(3-41); Arg^(26,34)Lys⁴²GLP-1(3-42);Arg^(26,34)Lys⁴³GLP-1(3-43); Arg^(26,34)Lys⁴⁴GLP-1(3-44);Arg^(26,34)Lys⁴⁵GLP-1(3-45); Arg^(26,34)Lys³⁸GLP-1(4-38);Arg^(26,34)Lys³⁹GLP-1(4-39); Arg^(26,34)Lys⁴⁰GLP-1(4-40);Arg^(26,34)Lys⁴¹GLP-1(4-41); Arg^(26,34)Lys⁴²GLP-1(4-42);Arg^(26,34)Lys⁴³GLP-1(4-43); Arg^(26,34)Lys⁴⁴GLP-1(4-44);Arg^(26,34)Lys⁴⁵GLP-1(4-45); Arg^(26,34)Lys³⁸GLP-1(5-38);Arg^(26,34)Lys³⁹GLP-1(5-39); Arg^(26,34)Lys⁴⁰GLP-1(5-40);Arg^(26,34)Lys⁴¹GLP-1(5-41); Arg^(26,34)Lys⁴²GLP-1(5-42);Arg^(26,34)Lys⁴³GLP-1(5-43); Arg^(26,34)Lys⁴⁴GLP-1(5-44);Arg^(26,34)Lys⁴⁵GLP-1(5-45); Arg^(26,34)Lys³⁸GLP-1(6-38);Arg^(26,34)Lys³⁹GLP-1(6-39); Arg^(26,34)Lys⁴⁰GLP-1(6-40);Arg^(26,34)Lys⁴¹GLP-1(6-41); Arg^(26,34)Lys⁴²GLP-1(6-42);Arg^(26,34)Lys⁴³GLP-1(6-43); Arg^(26,34)Lys⁴⁴GLP-1(6-44);Arg^(26,34)Lys⁴⁵GLP-1(6-45); Arg²⁶Lys³⁸GLP-1(1-38);Arg³⁴Lys³⁸GLP-1(1-38); Arg^(26,34)Lys^(36,38)GLP-1(1-38);Arg²⁶Lys³⁸GLP-1(7-38); Arg³⁴Lys³⁸GLP-1(7-38);Arg^(26,34)Lys^(36,38)GLP-1(7-38); Arg^(26,34)Lys³⁸GLP-1(7-38);Arg²⁶Lys³⁹GLP-1(1-39); Arg³⁴Lys³⁹GLP-1(1-39);Arg^(26,34)Lys^(36,39)GLP-1(1-39); Arg²⁶Lys³⁹GLP-1(7-39);Arg³⁴Lys³⁹GLP-1(7-39); Arg^(26,34)Lys^(36,39)GLP-1(7-39);Arg²⁶-GLP-1(8-37); Arg³⁴-GLP-1(8-37); Lys³⁶-GLP-1(8-37);Arg^(26,34)Lys³⁶-GLP-1(8-37); Arg^(26,34)Lys³⁸GLP-1(8-38);Arg^(26,34)Lys³⁹-GLP-1(8-39); Arg^(26,34)Lys⁴⁰-GLP-1(8-40);Arg²⁶Lys³⁶-GLP-1(8-37); Arg³⁴Lys³⁶-GLP-1(8-37); Arg²⁶Lys³⁹-GLP-1(8-39);Arg³⁴Lys⁴⁰-GLP-1(8-40); Arg^(26,34)Lys^(36,39)-GLP-1(8-39);Arg^(26,34)Lys^(36,40)-GLP-1(8-40); Gly⁸Arg²⁶-GLP-1(8-37);Gly⁸Arg³⁴-GLP-1(8-37); Gly⁸Lys³⁶-GLP-1(8-37);Gly⁸Arg^(26,34)Lys³⁶-GLP-1(8-37); Gly⁸Arg^(26,34)Lys³⁹-GLP-1(8-39);Gly⁸Arg^(26,34)Lys⁴⁰-GLP-1(8-40); Gly⁸Arg²⁶Lys³⁶-GLP-1(8-37);Gly⁸Arg³⁴Lys³⁶-GLP-1(8-37); Gly⁸Arg²⁶Lys³⁹-GLP-1(8-39);Gly⁸Arg³⁴Lys⁴⁰-GLP-1(8-40); Gly⁸Arg^(26,34)Lys^(36,39)-GLP-1(8-39);Gly⁸Arg^(26,34)Lys^(36,40)-GLP-1(8-40); Arg^(26,34)Lys³⁸GLP-1(8-38);Arg^(26,34)Lys³⁹GLP-1(8-39); Arg^(26,34)Lys⁴⁰GLP-1(8-40);Arg^(26,34)Lys⁴¹GLP-1(8-41); Arg^(26,34)Lys⁴²GLP-1(8-42);Arg^(26,34)Lys⁴³GLP-1(8-43); Arg^(26,34)Lys⁴⁴GLP-1(8-44);Arg^(26,34)Lys⁴⁵GLP-1(8-45); Arg²⁶Lys³⁸GLP-1(8-38);Arg³⁴Lys³⁸GLP-1(8-38); Arg^(26,34)Lys^(36,38)GLP-1(8-38);Arg^(26,34)Lys³⁸GLP-1(8-38); Arg²⁶Lys³⁹GLP-1(8-39);Arg³⁴Lys³⁹GLP-1(8-39); Arg^(26,34)Lys^(36,39)GLP-1(8-39);Arg²⁶-GLP-1(8-37), Arg³⁴-GLP-1(8-37), Lys³⁶-GLP-1(8-37),Arg^(26,34)Lys³⁶-GLP-1(8-37), Arg²⁶Lys³⁶-GLP-1(8-37), Arg³⁴-GLP-1(8-37),Gly⁸Arg²⁶-GLP-1(8-37), Gly⁸Arg³⁴-GLP-1(8-37), Gly⁸Lys³⁶-GLP-1(8-37),Gly⁸Arg^(26,34)Lys³⁶-GLP-1(8-37), Gly⁸Arg²⁶Lys³⁶-GLP-1(8-37),Gly⁸Arg³⁴Lys³⁶-GLP-1(8-37); Arg²⁶Lys³⁸-GLP-1(8-38),Arg^(26,34)Lys³⁸-GLP-1(8-38), Arg^(26,34)Lys^(36,38)-GLP-1(8-38),Gly⁸Arg²⁶Lys³⁸-GLP-1(8-38); Gly⁸Arg^(26,34)Lys^(36,38)-GLP-1(8-38);Arg³⁴Lys⁴⁰-GLP-1(8-40), Arg^(26,34)Lys^(36,40)-GLP-1(8-40),Gly⁸Arg³⁴Lys⁴⁰-GLP-1(8-40); Gly⁸Arg^(26,34)Lys^(36,40)-GLP-1(8-40);Arg²⁶-GLP-1(8-36); Arg³⁴-GLP-1(8-36); Arg^(26,34)Lys³⁶-GLP-1(8-36);Arg²⁶-GLP-1(8-36)amide; Arg³⁴-GLP-1(8-36)amide;Arg^(26,34)Lys³⁶-GLP-1(8-36)amide; Arg²⁶-GLP-1(8-37); Arg³⁴-GLP-1(8-37);Arg^(26,34)Lys³⁶-GLP-1(8-37); Arg²⁶-GLP-1(8-38); Arg³⁴GLP-1(8-38);Arg^(26,34)Lys³⁸GLP-1(8-38); Arg²⁶-GLP-1(8-39); Arg³⁴-GLP-1(8-39);Arg^(26,34)Lys³⁹-GLP-1(8-39); Gly⁸Arg²⁶-GLP-1(8-36);Gly⁸Arg³⁴-GLP-1(8-36); Gly⁸Arg^(26,34)Lys³⁶-GLP-1(8-36);Gly⁸Arg²⁶-GLP-1(8-36)amide; Gly⁸Arg³⁴-GLP-1(8-36)amide;Gly⁸Arg^(26,34)Lys³⁶-GLP-1(8-36)amide; Gly⁸Arg²⁶-GLP-1(8-37);Gly⁸Arg³⁴-GLP-1(8-37); Gly⁸Arg^(26,34)Lys³⁶-GLP-1(8-37);Gly⁸Arg²⁶-GLP-1(8-38); Gly⁸Arg³⁴-GLP-1(8-38);Gly⁸Arg^(26,34)Lys³⁸GLP-1(8-38); Gly⁸Arg²⁶-GLP-1(8-39);Gly⁸Arg³⁴-GLP-1(8-39); Gly⁸Arg^(26,34)Lys³⁹-GLP-1(8-39);Val⁸Arg²⁶-GLP-1(8-36); Val⁸Arg³⁴-GLP-1(8-36);Val⁸Arg^(26,34)Lys³⁶-GLP-1(8-36); Val⁸Arg²⁶-GLP-1(8-36)amide;Val⁸Arg³⁴-GLP-1(8-36)amide; Val⁸Arg^(26,34)Lys³⁶-GLP-1(8-36)amide;Val⁸Arg²⁶-GLP-1(8-37); Val⁸Arg³⁴-GLP-1(8-37);Val⁸Arg^(26,34)Lys³⁶-GLP-1(8-37); Val⁸Arg²⁶-GLP-1(8-38);Val⁸Arg³⁴-GLP-1(8-38); Val⁸Arg^(26,34)Lys³⁸GLP-1(8-38);Val⁸Arg²⁶-GLP-1(8-39); Val⁸Arg³⁴GLP-1(8-39);Val⁸Arg^(26,34)Lys³⁹-GLP-1(8-39); Ser⁸Arg²⁶-GLP-1(8-36);Ser⁸Arg³⁴-GLP-1(8-36); Ser⁸Arg^(26,43)Lys³⁶-GLP-1(8-36);Ser⁸Arg²⁶-GLP-1(8-36)amide; Ser⁸Arg³⁴-GLP-1(8-36)amide;Ser⁸Arg^(26,34)Lys³⁶-GLP-1(8-36)amide; Ser⁸Arg²⁶-GLP-1(8-37);Ser⁸Arg³⁴-GLP-1(8-37); Ser⁸Arg^(26,34)Lys³⁶-GLP-1(8-37);Ser⁸Arg²⁶-GLP-1(8-38); Ser⁸Arg³⁴-GLP-1(8-38);Ser⁸Arg^(26,34)Lys³⁸GLP-1(8-38); Ser⁸Arg²⁶-GLP-1(8-39);Ser⁸Arg³⁴-GLP-1(8-39); Ser⁸Arg^(26,34)Lys³⁹-GLP-1(8-39);Thr⁸Arg²⁶-GLP-1(8-36); Thr⁸Arg³⁴-GLP-1(8-36);Thr⁸Arg^(26,34)Lys³⁶-GLP-1(8-36); Thr⁸Arg²⁶-GLP-1(8-36)amide;Thr⁸Arg³⁴-GLP-1(8-36)amide; Thr⁸Arg^(26,34)Lys³⁶-GLP-1(8-36)amide;Thr⁸Arg²⁶-GLP-1(8-37); Thr⁸Arg³⁴-GLP-1(8-37);Thr⁸Arg^(26,34)Lys³⁶-GLP-1(8-37); Thr⁸Arg²⁶-GLP-1(8-38);Thr⁸Arg³⁴-GLP-1(8-38); Thr⁸Arg^(26,34)Lys³⁸GLP-1(8-38);Thr⁸Arg²⁶-GLP-1(8-39); Thr⁸Arg³⁴-GLP-1(8-39);Thr⁸Arg^(26,34)Lys³⁹-GLP-1(8-39); Val⁸Glu³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36); Val⁸Glu³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36)amide;Val⁸Glu³⁶Arg^(26,34)Lys³⁷GLP-1(8-37);Val⁸Glu³⁷Arg^(26,34)Lys³⁸GLP-1(8-38);Val⁸Glu³⁸Arg^(26,34)Lys³⁹-GLP-1(8-39);Val⁸Glu³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36);Val⁸Glu³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36)amide;Val⁸Glu³⁶Arg^(26,34)Lys³⁷GLP-1(8-37);Val⁸Glu³⁷Arg^(26,34)Lys³⁸GLP-1(8-38);Val⁸Glu³⁸Arg^(26,34)Lys³⁹GLP-1(8-39);Val⁸Asp³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36);Val⁸Asp³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36)amide;Val⁸Asp³⁶Arg^(26,34)Lys³⁷GLP-1(8-37);Val⁸Asp³⁷Arg^(26,34)Lys³⁸GLP-1(8-38);Val⁸Asp³⁸Arg^(26,34)Lys³⁹-GLP-1(8-39);Val⁸Asp³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36);Val⁸Asp³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36)amide;Val⁸Asp³⁶Arg^(26,34)Lys³⁷GLP-1(8-37);Val⁸Asp³⁷Arg^(26,34)Lys³⁸GLP-1(8-38);Val⁸Asp³⁸Arg^(26,34)Lys³⁹-GLP-1(8-39);Ser⁸Glu³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36);Ser⁸Glu³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36)amide;Ser⁸Glu³⁶Arg^(26,34)Lys³⁷GLP-1(8-37);Ser⁸Glu³⁷Arg^(26,34)Lys³⁸GLP-1(8-38);Ser⁸Glu³⁸Arg^(26,34)Lys³⁹-GLP-1(8-39);Ser⁸Glu³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36);Ser⁸Glu³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36)amide;Ser⁸Glu³⁶Arg^(26,34)Lys³⁷GLP-1(8-37);Ser⁸Glu³⁷Arg^(26,34)Lys³⁸GLP-1(8-38);Ser⁸Glu³⁸Arg^(26,34)Lys³⁹-GLP-1(8-39);Ser⁸Asp³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36);Ser⁸Asp³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36)amide;Ser⁸Asp³⁶Arg^(26,34)Lys³⁷GLP-1(8-37);Ser⁸Asp³⁷Arg^(26,34)Lys³⁸GLP-1(8-38);Ser⁸Asp³⁸Arg^(26,34)Lys³⁹-GLP-1(8-39);Ser⁸Asp³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36);Ser⁸Asp³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36)amide;Ser⁸Asp³⁶Arg^(26,34)Lys³⁷GLP-1(8-37);Ser⁸Asp³⁷Arg^(26,34)Lys³⁸GLP-1(8-38);Ser⁸Asp³⁸Arg^(26,34)Lys³⁹-GLP-1(8-39);Thr⁸Glu³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36);Thr⁸Glu³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36)amide;Thr⁸Glu³⁶Arg^(26,34)Lys³⁷GLP-1(8-37);Thr⁸Glu³⁷Arg^(26,34)Lys³⁸GLP-1(8-38);Thr⁸Glu³⁸Arg^(26,34)Lys³⁹-GLP-1(8-39);Thr⁸Glu³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36);Thr⁸Glu³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36)amide;Thr⁸Glu³⁶Arg^(26,34)Lys³⁷GLP-1(8-37);Thr⁸Glu³⁷Arg^(26,34)Lys³⁸GLP-1(8-38);Thr⁸Glu³⁸Arg^(26,34)Lys³⁹-GLP-1(8-39); Thr⁸Asp³⁵Arg^(26,34)Lys³⁶GLP-1(8-36); Thr⁸Asp³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36)amide;Thr⁸Asp³⁶Arg^(26,34)Lys³⁷GLP-1(8-37);Thr⁸Asp³⁷Arg^(26,34)Lys³⁸GLP-1(8-38);Thr⁸Asp³⁸Arg^(26,34)Lys³⁹-GLP-1(8-39);Thr⁸Asp³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36);Thr⁸Asp³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36)amide;Thr⁸Asp³⁶Arg^(26,34)Lys³⁷GLP-1(8-37);Thr⁸Asp³⁷Arg^(26,34)Lys³⁸GLP-1(8-38);Thr⁸Asp³⁸Arg^(26,34)Lys³⁹GLP-1(8-39);Gly⁸Glu³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36);Gly⁸Glu³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36)amide;Gly⁸Glu³⁶Arg^(26,34)Lys³⁷GLP-1(8-37);Gly⁸Glu³⁷Arg^(26,34)Lys³⁸GLP-1(8-38);Gly⁸Glu³⁸Arg^(26,34)Lys³⁹-GLP-1(8-39);Gly⁸Asp³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36);Gly⁸Glu³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36)amide;Gly⁸Glu³⁶Arg^(26,34)Lys³⁷GLP-1(8-37);Gly⁸Glu³⁷Arg^(26,34)Lys³⁸GLP-1(8-38);Gly⁸Glu³⁸Arg^(26,34)Lys³⁹-GLP-1(8-39);Gly⁸Asp³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36);Gly⁸Asp³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36)amide;Gly⁸Asp³⁶Arg^(26,34)Lys³⁷GLP-1(8-37);Gly⁸Asp³⁷Arg^(26,34)Lys³⁸GLP-1(8-38);Gly⁸Asp³⁸Arg^(26,34)Lys³⁹-GLP-1(8-39);Gly⁸Asp³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36);Gly⁸Asp³⁵Arg^(26,34)Lys³⁶-GLP-1(8-36)amide;Gly⁸Asp³⁶Arg^(26,34)Lys³⁷GLP-1(8-37);Gly⁸Asp³⁷Arg^(26,34)Lys³⁸GLP-1(8-38);Gly⁸Asp³⁸Arg^(26,34)Lys³⁹-GLP-1(8-39); Arg^(26,34)Lys¹⁸-GLP-1(8-36);Arg^(26,34)Lys¹⁸-GLP-1(8-36)amide; Arg^(26,34)Lys¹⁸GLP-1(8-37);Arg^(26,34)Lys¹⁸GLP-1(8-38); Gly⁸Asp¹⁹Arg^(26,34)Lys¹⁸-GLP-1(8-36);Gly⁸Asp¹⁷Arg^(26,34)Lys¹⁸-GLP-1(8-36);Gly⁸Asp¹⁹Arg^(26,34)Lys¹⁸-GLP-1(8-36)amide;Gly⁸Asp¹⁷Arg^(26,34)Lys¹⁸-GLP-1(8-36)amide;Gly⁸Asp¹⁹Arg^(26,34)Lys¹⁸GLP-1(8-37);Gly⁸Asp¹⁹Arg^(26,34)Lys¹⁸GLP-1(8-38);Gly⁸Asp¹⁷Arg^(26,34)Lys¹⁸GLP-1(8-38); Arg^(26,34)Lys²³GLP-1(8-36);Arg^(26,34)Lys²³GLP-1(8-36)amide; Arg^(26,34)Lys²³GLP-1(8-37);Arg^(26,34)Lys²³GLP-1(8-38); Gly⁸Asp²⁴Arg^(26,34)Lys²³-GLP-1(8-36);Gly⁸Asp²²Arg^(26,34)Lys²³-GLP-1(8-36);Gly⁸Asp²⁴Arg^(26,34)Lys²³-GLP-1(8-36)amide;Gly⁸Asp²²Arg^(26,34)Lys²³-GLP-1(8-36)amide;Gly⁸Asp²⁴Arg^(26,34)Lys²³GLP-1(8-37);Gly⁸Asp²⁴Arg^(26,34)Lys²³GLP-1(8-38);Gly⁸Asp²²Arg^(26,34)Lys²³GLP-1(8-38); Arg^(26,34)Lys²⁷-GLP-1(8-36);Arg^(26,34)Lys²⁷-GLP-1(8-36)amide; Arg^(26,34)Lys²⁷GLP-1(8-37);Arg^(26,34)Lys²⁷GLP-1(8-38); Gly⁸Asp²⁸Arg^(26,34)Lys²⁷-GLP-1(8-36);Gly⁸Asp²⁶Arg^(26,34)Lys²⁷-GLP-1(8-36);Gly⁸Asp²⁸Arg^(26,34)Lys²⁷-GLP-1(8-36)amide;Gly⁸Asp²⁶Arg^(26,34)Lys²⁷-GLP-1(8-36)amide;Gly⁸Asp²⁸Arg^(26,34)Lys²⁷GLP-1(8-37);Gly⁸Asp²⁸Arg^(26,34)Lys²⁷GLP-1(8-38);Gly⁸Asp²⁶Arg^(26,34)Lys²⁷GLP-1(8-38); Arg^(26,34)Lys¹⁸-GLP-1(8-36);Arg^(26,34)Lys¹⁸-GLP-1(8-36)amide; Arg^(26,34)Lys¹⁸-GLP-1(8-37);Arg^(26,34)Lys¹⁸GLP-1(8-38); Val⁸Asp¹⁹Arg^(26,34)Lys¹⁸-GLP-1(8-36);Val⁸Asp¹⁷Arg^(26,34)Lys¹⁸-GLP-1(8-36);Val⁸Asp¹⁹Arg^(26,34)Lys¹⁸-GLP-1(8-36)amide;Val⁸Asp¹⁷Arg^(26,34)Lys¹⁸-GLP-1(8-36)amide;Val⁸Asp¹⁹Arg^(26,34)Lys¹⁸GLP-1(8-37);Val⁸Asp¹⁹Arg^(26,34)Lys¹⁸GLP-1(8-38);Val⁸Asp¹⁷Arg^(26,34)Lys¹⁸GLP-1(8-38); Arg^(26,34)Lys²³-GLP-1(8-36);Arg^(26,34)Lys²³-GLP-1(8-36)amide; Arg^(26,34)Lys²³GLP-1(8-37);Arg^(26,34)Lys²³GLP-1(8-38); Val⁸Asp²⁴Arg^(26,34)Lys²³-GLP-1(8-36);Val⁸Asp²²Arg^(26,34)Lys²³-GLP-1(8-36);Val⁸Asp²⁴Arg^(26,34)Lys²³-GLP-1(8-36)amide;Val⁸Asp²²Arg^(26,34)Lys²³-GLP-1(8-36)amide;Val⁸Asp²⁴Arg^(26,34)Lys²³GLP-1(8-37);Val⁸Asp²⁴Arg^(26,34)Lys²³GLP-1(8-38);Val⁸Asp²²Arg^(26,34)Lys²³GLP-1(8-38); Arg^(26,34)Lys²⁷-GLP-1(8-36);Arg^(26,34)Lys²⁷-GLP-1(8-36)amide; Arg^(26,34)Lys²⁷GLP-1(8-37);Arg^(26,34)Lys²⁷GLP-1(8-38); Val⁸Asp²⁸Arg^(26,34)Lys²⁷-GLP-1(8-36);Val⁸Asp²⁶Arg^(26,34)Lys²⁷-GLP-1(8-36);Val⁸Asp²⁸Arg^(26,34)Lys²⁷-GLP-1(8-36)amide;Val⁸Asp²⁶Arg^(26,34)Lys²⁷GLP-1(8-36)amide;Val⁸Asp²⁸Arg^(26,34)Lys²⁷GLP-1(8-37);Val⁸Asp²⁸Arg^(26,34)Lys²⁷GLP-1(8-38);Val⁸Asp²⁶Arg^(26,34)Lys²⁷GLP-1(8-38); Arg^(26,34)Lys¹⁸-GLP-1(8-36);Arg^(26,34)Lys¹⁸-GLP-1(8-36)amide; Arg^(26,34)Lys¹⁸GLP-1(8-37);Arg^(26,34)Lys¹⁸GLP-1(8-38); Ser⁸Asp¹⁹Arg^(26,34)Lys¹⁸-GLP-1(8-36);Ser⁸Asp¹⁷Arg^(26,34)Lys¹⁸-GLP-1(8-36);Ser⁸Asp¹⁹Arg^(26,34)Lys¹⁸-GLP-1(8-36)amide;Ser⁸Asp¹⁷Arg^(26,34)Lys¹⁸-GLP-1(8-36)amide;Ser⁸Asp¹⁹Arg^(26,34)Lys¹⁸GLP-1(8-37);Ser⁸Asp¹⁹Arg^(26,34)Lys¹⁸GLP-1(8-38);Ser⁸Asp¹⁷Arg^(26,34)Lys¹⁸GLP-1(8-38); Arg^(26,34)Lys²³-GLP-1(8-36);Arg^(26,34)Lys²³-GLP-1(8-36)amide; Arg^(26,34)Lys²³GLP-1(8-37);Arg^(26,34)Lys²³GLP-1(8-38); Ser⁸Asp²⁴Arg^(26,34)Lys²³-GLP-1(8-36);Ser⁸Asp²²Arg^(26,34)Lys²³-GLP-1(8-36);Ser⁸Asp²⁴Arg^(26,34)Lys²³-GLP-1(8-36)amide;Ser⁸Asp²²Arg^(26,34)Lys²³-GLP-1(8-36)amide;Ser⁸Asp²⁴Arg^(26,34)Lys²³GLP-1(8-37);Ser⁸Asp²⁴Arg^(26,34)Lys²³GLP-1(8-38);Ser⁸Asp²²Arg^(26,34)Lys²³GLP-1(8-38); Arg^(26,34)Lys²⁷-GLP-1(8-36);Arg^(26,34)Lys²⁷-GLP-1(8-36)amide; Arg^(26,34)Lys²⁷-GLP-1(8-37);Arg^(26,34)Lys²⁷GLP-1(8-38); Ser⁸Asp²⁸Arg^(26,34)Lys²⁷-GLP-1(8-36);Ser⁸Asp²⁶Arg^(26,34)Lys²⁷GLP-1(8-36);Ser⁸Asp²⁸Arg^(26,34)Lys²⁷-GLP-1(8-36)amide;Ser⁸Asp²⁶Arg^(26,34)Lys²⁷-GLP-1(8-36)amide;Ser⁸Asp²⁸Arg^(26,34)Lys²⁷GLP-1(8-37);Ser⁸Asp²⁸Arg^(26,34)Lys²⁷GLP-1(8-38);Ser⁸Asp²⁶Arg^(26,34)Lys²⁷GLP-1(8-38); Arg^(26,34)Lys¹⁸-GLP-1(8-36);Arg^(26,34)Lys¹⁸-GLP-1(8-36)amide; Arg^(26,34)Lys¹⁸GLP-1(8-37);Arg^(26,34)Lys¹⁸GLP-1(8-38); Thr⁸Asp¹⁹Arg^(26,34)Lys¹⁸-GLP-1(8-36);Thr⁸Asp¹⁷Arg^(26,34)Lys¹⁸-GLP-1(8-36);Thr⁸Asp¹⁹Arg^(26,34)Lys¹⁸-GLP-1(8-36)amide;Thr⁸Asp¹⁷Arg^(26,34)Lys¹⁸-GLP-1(8-36)amide;Thr⁸Asp¹⁹Arg^(26,34)Lys¹⁸GLP-1(8-37);Thr⁸Asp¹⁹Arg^(26,34)Lys¹⁸GLP-1(8-38);Thr⁸Asp¹⁷Arg^(26,34)Lys¹⁸GLP-1(8-38); Arg^(26,34)Lys²³-GLP-1(8-36);Arg^(26,34)Lys²³-GLP-1(8-36)amide; Arg^(26,34)Lys²³GLP-1(8-37);Arg^(26,34)Lys²³GLP-1(8-38); Thr⁸Asp²⁴Arg^(26,34)Lys²³-GLP-1(8-36);Thr⁸Asp²²Arg^(26,34)Lys²³-GLP-1(8-36);Thr⁸Asp²⁴Arg^(26,34)Lys²³-GLP-1(8-36)amide;Thr⁸Asp²²Arg^(26,34)Lys²³-GLP-1(8-36)amide;Thr⁸Asp²⁴Arg^(26,34)Lys²³GLP-1(8-37);Thr⁸Asp²⁴Arg^(26,34)Lys²³GLP-1(8-38);Thr⁸Asp²²Arg^(26,34)Lys²³GLP-1(8-38); Arg^(26,34)Lys²⁷-GLP-1(8-36);Arg^(26,34)Lys²⁷-GLP-1(8-36)amide; Arg^(26,34)Lys²⁷GLP-1(8-37);Arg^(26,34)Lys²⁷GLP-1(8-38); Thr⁸Asp²⁸Arg^(26,34)Lys²⁷-GLP-1(8-36);Thr⁸Asp²⁶Arg^(26,34)Lys²⁷-GLP-1(8-36);Thr⁸Asp²⁸Arg^(26,34)Lys²⁷-GLP-1(8-36)amide;Thr⁸Asp²⁶Arg^(26,34)Lys²⁷-GLP-1(8-36)amide;Thr⁸Asp²⁸Arg^(26,34)Lys²⁷GLP-1(8-37);Thr⁸Asp²⁸Arg^(26,34)Lys²⁷GLP-1(8-38);Thr⁸Asp²⁶Arg^(26,34)Lys²⁷GLP-1(8-38); Arg²⁶Lys³⁶-GLP-1(8-36);Arg³⁴Lys³⁶-GLP-1(8-36); Arg²⁶Lys³⁶-GLP-1(8-37); Arg³⁴Lys³⁶-GLP-1(8-37);Arg²⁶Lys³⁷-GLP-1(8-37); Arg³⁴Lys³⁷-GLP-1(8-37); Arg²⁶Lys³⁹-GLP-1(8-39);Arg³⁴Lys³⁹-GLP-1(8-39); Arg^(26,34)Lys^(36,39)-GLP-1(8-39);Arg²⁶Lys¹⁸-GLP-1(8-36); Arg³⁴Lys¹⁸-GLP-1(8-36); Arg²⁶Lys¹⁸GLP-1(8-37);Arg³⁴Lys¹⁸GLP-1(8-37); Arg²⁶Lys¹⁸GLP-1(8-38); Arg³⁴Lys¹⁸GLP-1(8-38);Arg²⁶Lys¹⁸GLP-1(8-39); Arg³⁴Lys¹⁸GLP-1(8-39); Arg²⁶Lys²³-GLP-1(8-36);Arg³⁴Lys²³-GLP-1(8-36); Arg²⁶Lys²³GLP-1(8-37); Arg³⁴Lys²³GLP-1(8-37);Arg²⁶Lys²³GLP-1(8-38); Arg³⁴Lys²³GLP-1(8-38); Arg²⁶Lys²³GLP-1(8-39);Arg³⁴Lys²³GLP-1(8-39); Arg²⁶Lys²⁷-GLP-1(8-36); Arg³⁴Lys²⁷-GLP-1(8-36);Arg²⁶Lys²⁷GLP-1(8-37); Arg³⁴Lys²⁷GLP-1(8-37); Arg²⁶Lys²⁷GLP-1(8-38);Arg³⁴Lys²⁷GLP-1(8-38); Arg²⁶Lys²⁷GLP-1(8-39); Arg³⁴Lys²⁷GLP-1(8-39);Arg^(26,34)Lys^(18,36)-GLP-1(8-36); Arg^(26,34)Lys¹⁸GLP-1(8-37);Arg^(26,34)Lys^(18,37)GLP-1(8-37); Arg^(26,34)Lys^(18,38)GLP-1(8-38);Arg^(26,34)Lys^(18,39)GLP-1(8-39); Arg^(26,34)Lys^(23,36)-GLP-1(8-36);Arg^(26,34)Lys²³GLP-1(8-37); Arg^(26,34)Lys^(23,37)GLP-1(8-37);Arg^(26,34)Lys^(23,38)GLP-1(8-38); Arg^(26,34)Lys^(23,39)GLP-1(8-39);Arg^(26,34)Lys^(27,36)-GLP-1(8-36); Arg^(26,34)Lys^(27,)-GLP-1(8-37);Arg^(26,34)Lys^(27,37)GLP-1(8-37); Arg^(26,34)Lys^(27,38)GLP-1(8-38);Arg^(26,34)Lys^(27,39)GLP-1(8-39); Gly⁸GLP-1(8-36); Gly⁸GLP-1(8-37);Gly⁸GLP-1(8-38); Gly⁸GLP-1(8-39); Gly⁸Arg²⁶Lys³⁶-GLP-1(8-36);Gly⁸Arg³⁴Lys³⁶-GLP-1(8-36); Gly⁸Arg²⁶Lys³⁶-GLP-1(8-37);Gly⁸Arg³⁴Lys³⁶-GLP-1(8-37); Gly⁸Arg²⁶Lys³⁷-GLP-1(8-37);Gly⁸Arg³⁴Lys³⁷-GLP-1(8-37); Gly⁸Arg²⁶Lys³⁹-GLP-1(8-39);Gly⁸Arg³⁴Lys³⁹-GLP-1(8-39); Gly⁸Arg^(26,34)Lys^(36,39)-GLP-1(8-39);Gly⁸Arg²⁶Lys¹⁸-GLP-1(8-36); Gly⁸Arg³⁴Lys¹⁸-GLP-1(8-36);Gly⁸Arg²⁶Lys¹⁸GLP-1(8-37); Gly⁸Arg³⁴Lys¹⁸GLP-1(8-37);Gly⁸Arg²⁶Lys¹⁸GLP-1(8-38); Gly⁸Arg³⁴Lys¹⁸GLP-1(8-38);Gly⁸Arg²⁶Lys¹⁸GLP-1(8-39); Gly⁸Arg³⁴Lys¹⁸GLP-1(8-39);Gly⁸Arg²⁶Lys²³-GLP-1(8-36); Gly⁸Arg³⁴Lys²³-GLP-1(8-36);Gly⁸Arg²⁶Lys²³GLP-1(8-37); Gly⁸Arg³⁴Lys²³GLP-1(8-37);Gly⁸Arg²⁶Lys²³GLP-1(8-38); Gly⁸Arg³⁴Lys²³GLP-1(8-38);Gly⁸Arg²⁶Lys²³GLP-1(8-39); Gly⁸Arg³⁴Lys²³GLP-1(8-39);Gly⁸Arg²⁶Lys²⁷-GLP-1(8-36); Gly⁸Arg³⁴Lys²⁷-GLP-1(8-36);Gly⁸Arg²⁶Lys²⁷GLP-1(8-37); Gly⁸Arg³⁴Lys²⁷GLP-1(8-37);Gly⁸Arg²⁶Lys²⁷GLP-1(8-38); Gly⁸Arg³⁴Lys²⁷GLP-1(8-38);Gly⁸Arg²⁶Lys²⁷GLP-1(8-39); Gly⁸Arg³⁴Lys²⁷GLP-1(8-39);Gly⁸Arg^(26,34)Lys^(18,36)-GLP-1(8-36); Gly⁸Arg^(26,34)Lys¹⁸GLP-1(8-37);Gly⁸Arg^(26,34)Lys^(18,37)GLP-1(8-37);Gly⁸Arg^(26,34)Lys^(18,38)GLP-1(8-38);Gly⁸Arg^(26,34)Lys^(18,39)GLP-1(8-39);Gly⁸Arg^(26,34)Lys^(23,36)-GLP-1(8-36); Gly⁸Arg^(26,34)Lys²³GLP-1(8-37);Gly⁸Arg^(26,34)Lys^(23,37)GLP-1(8-37);Gly⁸Arg^(26,34)Lys^(23,38)GLP-1(8-38);Gly⁸Arg^(26,34)Lys^(23,39)GLP-1(8-39);Gly⁸Arg^(26,34)Lys^(27,36)-GLP-1(8-36); Gly⁸Arg^(26,34)Lys²⁷GLP-1(8-37);Gly⁸Arg^(26,34)Lys^(27,37)GLP-1(8-37);Gly⁸Arg^(26,34)Lys^(27,38)GLP-1(8-38);Gly⁸Arg^(26,34)Lys^(27,39)GLP-1(8-39); Val⁸GLP-1(8-36); Val⁸GLP-1(8-37);Val⁸GLP-1(8-38); Val⁸GLP-1(8-39) Val⁸Arg²⁶Lys³⁶-GLP-1(8-36);Val⁸Arg³⁴Lys³⁶-GLP-1(7-36); Val⁸Arg²⁶Lys³⁶-GLP-1(8-37);Val⁸Arg³⁴Lys³⁶-GLP-1(8-37); Val⁸Arg²⁶Lys³⁷-GLP-1(8-37);Val⁸Arg³⁴Lys³⁷-GLP-1(8-37); Val⁸Arg²⁶Lys³⁹-GLP-1(8-39);Val⁸Arg³⁴Lys³⁹-GLP-1(8-39); Val⁸Arg^(26,34)Lys^(36,39)-GLP-1(8-39);Val⁸Arg²⁶Lys¹⁸-GLP-1(8-36); Val⁸Arg³⁴Lys¹⁸-GLP-1(8-36);Val⁸Arg²⁶Lys¹⁸GLP-1(8-37); Val⁸Arg³⁴Lys¹⁸GLP-1(8-37);Val⁸Arg²⁶Lys¹⁸GLP-1(8-38); Val⁸Arg³⁴Lys¹⁸GLP-1(8-38);Val⁸Arg²⁶Lys¹⁸GLP-1(8-39); Val⁸Arg³⁴Lys¹⁸GLP-1(8-39);Val⁸Arg²⁶Lys²³-GLP-1(8-36);Val⁸Arg³⁴Lys²³-GLP-1(8-36);Val⁸Arg²⁶Lys²³GLP-1(8-37); Val⁸Arg³⁴Lys²³GLP-1(8-37);Val⁸Arg²⁶Lys²³GLP-1(8-38); Val⁸Arg³⁴Lys²³GLP-1(8-38);Val⁸Arg²⁶Lys²³GLP-1(8-39); Val⁸Arg³⁴Lys²³GLP-1(8-39);Val⁸Arg²⁶Lys²⁷-GLP-1(8-36); Val⁸Arg³⁴Lys²⁷-GLP-1(8-36);Val⁸Arg²⁶Lys²⁷GLP-1(8-37); Val⁸Arg³⁴Lys²⁷GLP-1(8-37);Val⁸Arg²⁶Lys²⁷GLP-1(8-38); Val⁸Arg³⁴Lys²⁷GLP-1(8-38);Val⁸Arg²⁶Lys²⁷GLP-1(8-39); Val⁸Arg³⁴Lys²⁷GLP-1(8-39);Val⁸Arg^(26,34)Lys^(18,36)-GLP-1(8-36); Val⁸Arg^(26,34)Lys¹⁸GLP-1(8-37);Val⁸Arg^(26,34)Lys^(18,37)GLP-1(8-37);Val⁸Arg^(26,34)Lys^(18,38)GLP-1(8-38);Val⁸Arg^(26,34)Lys^(18,39)GLP-1(8-39);Val⁸Arg^(26,34)Lys^(23,36)-GLP-1(8-36); Val⁸Arg^(26,34)Lys²³GLP-1(8-37);Val⁸Arg^(26,34)Lys^(23,37)GLP-1(8-37);Val⁸Arg^(26,34)Lys^(23,38)GLP-1(8-38);Val⁸Arg^(26,34)Lys^(23,39)GLP-1(8-39);Val⁸Arg^(26,34)Lys^(27,36)-GLP-1(8-36); Val⁸Arg^(26,34)Lys²⁷GLP-1(8-37);Val⁸Arg^(26,34)Lys^(27,37)GLP-1(8-37);Val⁸Arg^(26,34)Lys^(27,38)GLP-1(8-38);Val⁸Arg^(26,34)LYs^(27,39)GLP-1(8-39); Val⁸GLP-1(7-37); Thr⁸GLP-1(7-37);Met⁸GLP-1(7-37); Gly⁸GLP-1(7-37); Val⁸GLP-1(7-36) amide; Thr⁸GLP-1(7-36)amide; Met⁸GLP-1(7-36) amide; Gly⁸GLP-1(7-36) amide;Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-37);Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-37);Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-37);Gly⁸Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-37);Gly⁸Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-37);Gly⁸Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-37);Arg²⁶Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-37);Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-38);Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-38);Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-38);Gly⁸Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-38);Gly⁸Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-38);Gly⁸Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-38);Arg²⁶Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-38);Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-39);Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-39);Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-39);Gly⁸Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-39);Gly⁸Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-39);Gly⁸Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-39);Arg²⁶Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-39);Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-40);Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-40);Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-40);Gly⁸Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-40);Gly⁸Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-40);Gly⁸Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-40);Arg²⁶Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-40);Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-36);Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-36);Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-36);Gly⁸Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-36);Gly⁸Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-36);Gly⁸Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-36);Arg²⁶Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-36);Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-35);Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-35);Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-35);Gly⁸Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-35);Gly⁸Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-35);Gly⁸Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-35);Arg²⁶Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-35);Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-36)amide;Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-36)amide;Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-36)amide;Gly⁸Lys²⁶(N^(ε)-tetradecanoyl)-GLP-1(7-36)amide;Gly⁸Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-36)amide;Gly⁸Lys^(26,34)-bis(N^(ε)-tetradecanoyl)-GLP-1(7-36)amide;Arg²⁶Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-36)amide;Gly⁸Arg²⁶Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-37);Lys²⁶(N^(ε)-tetradecanoyl)Arg³⁴-GLP-1(7-37);Gly⁸Lys²⁶(N^(ε)-tetradecanoyl)Arg³⁴-GLP-1(7-37);Arg^(26,34)Lys³⁶(N^(ε)-tetradecanoyl)-GLP-1(7-37);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-tetradecanoyl)-GLP-1(7-37);Gly⁸Arg²⁶Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-38);Lys²⁶(N^(ε)-tetradecanoyl)Arg³⁴-GLP-1(7-38);Gly⁸Lys²⁶(N^(ε)-tetradecanoyl)Arg³⁴-GLP-1(7-38);Arg^(26,34)Lys³⁶(N^(ε)-tetradecanoyl)-GLP-1(7-38);Arg^(26,34)Lys³⁸(N^(ε)-tetradecanoyl)-GLP-1(7-38);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-tetradecanoyl)-GLP-1(7-38);Gly⁸Arg²⁶Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-39);Lys²⁶(N^(ε)-tetradecanoyl)Arg³⁴-GLP-1(7-39);Gly⁸Lys²⁶(N^(ε)-tetradecanoyl)Arg³⁴-GLP-1(7-39);Arg^(26,34)Lys³⁶(N^(ε)-tetradecanoyl)-GLP-1(7-39);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-tetradecanoyl)-GLP-1(7-39);Gly⁸Arg²⁶Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-40);Lys²⁶(N^(ε)-tetradecanoyl)Arg³⁴-GLP-1(7-40);Gly⁸Lys²⁶(N^(ε)-tetradecanoyl)Arg³⁴-GLP-1(7-40);Arg^(26,34)Lys³⁶(N^(ε)-tetradecanoyl)-GLP-1(7-40);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-tetradecanoyl)-GLP-1(7-40);Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-37);Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-37);Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-37);Gly⁸Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-37);Gly⁸Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-37);Gly⁸Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-37);Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-38);Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-38);Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-38);Gly⁸Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-38);Gly⁸Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-38);Gly⁸Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-38);Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-39);Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-39);Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-39);Gly⁸Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-39);Gly⁸Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-39);Gly⁸Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-39);Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-40);Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-40);Lys^(26,34)-bis(N^(ε)-(-ω-carboxynonadecanoyl))-GLP-1(7-40);Gly⁸Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-40);Gly⁸Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-40);Gly⁸Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-40);Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36);Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36);Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36);Gly⁸Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36);Gly⁸Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36);Gly⁸Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36);Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36)amide;Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36)amide;Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36)amide;Gly⁸Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36)amide;Gly⁸Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36)amide;Gly⁸Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-36)amide;Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-35);Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-35);Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-35);Gly⁸Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-35);Gly⁸Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-35);Gly⁸Lys^(26,34)-bis(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-35);Arg²⁶Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-37);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-37);Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))Arg³⁴-GLP-1(7-37);Gly⁸Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))Arg³⁴-GLP-1(7-37);Arg^(26,34)Lys³⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-37);Gly⁸Arg^(26,34)Lys⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-37);Arg²⁶Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-38);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-38);Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))Arg³⁴-GLP-1(7-38);Gly⁸Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))Arg³⁴-GLP-1(7-38);Arg^(26,34)Lys³⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-38);Arg^(26,34)Lys³⁸(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-38);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-38);Arg²⁶Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-39);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-39);Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))Arg³⁴-GLP-1(7-39);Gly⁸Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))Arg³⁴-GLP-1(7-39);Arg^(26,34)Lys³⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-39);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-39);Arg³⁴Lys²⁶(N^(ε)-(γ-glutamyl(N^(α)-hexadecanoyl)))-GLP-1(7-37)-OH,Lys^(26,34)-bis(N^(ε)-(γ-glutamyl(N^(α)-hexadecanoyl)))-GLP-1(7-37)-OH,Lys^(26,34)-bis(N^(ε)-(γ-glutamyl(N^(α)-tetradecanoyl)))-GLP-1(7-37)-OH,Arg^(26,34)Lys³⁸(N^(ε)-(γ-glutamyl(N^(α)-tetradecanoyl)))-GLP-1(7-38)-OH,Arg^(26,34)Lys³⁸(N^(ε)-(γ-glutamyl(N^(α)-hexadecanoyl)))-GLP-1(7-38)-OH,Arg²⁶Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-40);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-40);Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))Arg³⁴-GLP-1(7-40);Gly⁸Lys²⁶(N^(ε)-(ω-carboxynonadecanoyl))Arg³⁴-GLP-1(7-40);Arg^(26,34)Lys³⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-40);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(ω-carboxynonadecanoyl))-GLP-1(7-40);Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-37);Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-37);Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-37);Gly⁸Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-37);Gly⁸Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-37);Gly⁸Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-37);Arg²⁶Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-37);Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-38);Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-38);Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-38);Gly⁸Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-38);Gly⁸Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-38);Gly⁸Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-38);Arg²⁶Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-38);Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-39);Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-39);Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-39);Gly⁸Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-39);Gly⁸Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-39);Gly⁸Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-39);Arg²⁶Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-39);Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-40);Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-40);Lys^(26,34)-bis(N⁶-(7-deoxycholoyl))-GLP-1(7-40);Gly⁸Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-40);Gly⁸Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-40);Gly⁸Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-40);Arg²⁶Lys³⁴(N^(ε)-7-deoxycholoyl))-GLP-1(7-40);Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36);Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36);Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36);Gly⁸Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36);Gly⁸Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36);Gly⁸Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36);Arg²⁶Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36);Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-35);Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-35);Lys^(26,34)-bis(N⁸-(7-deoxycholoyl))-GLP-1(7-35);Gly⁸Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-35);Gly⁸Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-35);Gly⁸Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-35);Arg²⁶Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-35);Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36)amide;Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36)amide;Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36)amide;Gly⁸Lys²⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36)amide;Gly⁸Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36)amide;Gly⁸Lys^(26,34)-bis(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36)amide;Arg²⁶Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-36)amide;Gly⁸Arg²⁶Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-37);Lys²⁶(N^(ε)-(7-deoxycholoyl))Arg³⁴-GLP-1(7-37);Gly⁸Lys²⁶(N^(ε)-(7-deoxycholoyl))Arg³⁴-GLP-1(7-37);Arg^(26,34)Lys³⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-37);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-37);Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-37); Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-37);Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-37);Gly⁸Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-37);Gly⁸Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-37);Gly⁸Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-37);Arg²⁶Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-37);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-38);Lys²⁶(N^(ε)-(7-deoxycholoyl))Arg³⁴-GLP-1(7-38);Gly⁸Lys²⁶(N^(ε)-(7-deoxycholoyl))Arg³⁴GLP-1(7-38);Arg^(26,34)Lys³⁶(N^(ε)-(7-deoxycholoyl))GLP-1(7-38);Arg^(26,34)Lys³⁸(N^(ε)-(7-deoxycholoyl))-GLP-1(7-38);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-38);Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-38); Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-38);Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-38);Gly⁸Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-38);Gly⁸Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-38);Gly⁸Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-38);Arg²⁶Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-38);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-39);Lys²⁶(N^(ε)-(7-deoxycholoyl))Arg³⁴GLP-1(7-39);Gly⁸Lys²⁶(N^(ε)-(7-deoxycholoyl))Arg³⁴-GLP-1(7-39);Arg^(26,34)Lys³⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-39);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-39);Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-39); Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-39);Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-39);Gly⁸Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-39);Gly⁸Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-39);Gly⁸Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-39);Arg²⁶Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-39);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(7-deoxycholoyl))-GLP-1(7-40);Lys²⁶(N^(ε)-(7-deoxycholoyl))Arg³-GLP-1(7-40);Gly⁸Lys²⁶(N^(ε)-(7-deoxycholoyl))Arg³⁴GLP-1(7-40);Arg^(26,34)Lys³⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-40);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(7-deoxycholoyl))-GLP-1(7-40);Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-40); Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-40);Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-40);Gly⁸Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-40);Gly⁸Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-40);Gly⁸Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-40);Arg²⁶Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-40);Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-36); Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-36);Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-36);Gly⁸Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-36);Gly⁸Lys³⁴(N⁸-(choloyl))-GLP-1(7-36);Gly⁸Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-36);Arg²⁶Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-36);Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-35); Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-35);Lys^(26,34)-bis(N⁸-(choloyl))-GLP-1(7-35);Gly⁸Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-35);Gly⁸Lys³⁴(N⁸-(choloyl))-GLP-1(7-35);Gly⁸Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-35);Arg²⁶Lys³⁴(N^(ε)-choloyl))-GLP-1(7-35);Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-36)amide;Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-36)amide;Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-36)amide;Gly⁸Lys²⁶(N^(ε)-(choloyl))-GLP-1(7-36)amide;Gly⁸Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-36)amide;Gly⁸Lys^(26,34)-bis(N^(ε)-(choloyl))-GLP-1(7-36)amide;Arg²⁶Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-36)amide;Gly⁸Arg²⁶Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-37);Lys²⁶(N^(ε)-(choloyl))Arg³⁴-GLP-1(7-37);Gly⁸Lys²⁶(N^(ε)-(choloyl))Arg³⁴-GLP-1(7-37);Arg^(26,34)Lys³⁶(N^(ε)-(choloyl))-GLP-1(7-37);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(choloyl))-GLP-1(7-37);Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-37);Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-37);Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-37);Gly⁸Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-37);Gly⁸Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-37);Gly⁸Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-37);Arg²⁶Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-37);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-38);Lys²⁶(N^(ε)-(choloyl))Arg³⁴-GLP-1(7-38);Gly⁸Lys²⁶(N^(ε)-(choloyl))Arg³⁴-GLP-1(7-38);Arg^(26,34)Lys³⁶(N^(ε)-(choloyl))-GLP-1(7-38);Arg^(26,34)Lys³⁸(N^(ε)-(choloyl))-GLP-1(7-38);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(choloyl))-GLP-1(7-38);Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-38);Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-38);Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-38);Gly⁸Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-38);Gly⁸Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-38);Gly⁸Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-38);Arg²⁶Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-38);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-39);Lys²⁶(N⁸-(choloyl))Arg³⁴-GLP-1(7-39);Gly⁸Lys²⁶(N^(ε)-(choloyl))Arg³⁴-GLP-1(7-39);Arg^(26,34)Lys³⁶(N^(ε)-(choloyl))-GLP-1(7-39);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(choloyl))-GLP-1(7-39);Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-39);Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-39);Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-39);Gly⁸Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-39);Gly⁸Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-39);Gly⁸Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-39);Arg²⁶Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-39);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(choloyl))-GLP-1(7-40);Lys²⁶(N^(ε)-(choloyl))Arg³⁴-GLP-1(7-40); Gly⁸Lys²⁶(N^(ε)-(choloyl))Arg³⁴GLP-1(7-40); Arg^(26,34)Lys³⁶(N^(ε)-(choloyl))-GLP-1(7-40);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(choloyl))-GLP-1(7-40);Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-40);Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-40);Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-40);Gly⁸Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-40);Gly⁸Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-40);Gly⁸Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-40);Arg²⁶Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-37);Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-36);Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-36);Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-36);Gly⁸Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-36);Gly⁸Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-36);Gly⁸Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-36);Arg²⁶Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-36);Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-35);Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-35);Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-35);Gly⁸Lys²⁶(N^(ε)-lithocholoyl))-GLP-1(7-35);Gly⁸Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-35);Gly⁸Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-35);Arg²⁶Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-35);Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-36)amide;Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-36)amide;Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-36)amide;Gly⁸Lys²⁶(N^(ε)-(lithocholoyl))-GLP-1(7-36)amide;Gly⁸Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-36)amide;Gly⁸Lys^(26,34)-bis(N^(ε)-(lithocholoyl))-GLP-1(7-36)amide;Arg²⁶Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-36)amide;Gly⁸Arg²⁶Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-37);Lys²⁶(N^(ε)-(lithocholoyl))Arg³⁴GLP-1(7-37);Gly⁸Lys²⁶(N^(ε)-(lithocholoyl))Arg³⁴-GLP-1(7-37);Arg^(26,34)Lys³⁶(N^(ε)-(lithocholoyl))-GLP-1(7-37);Arg^(26,34)Lys³⁸(N^(ε)-(lithocholoyl))-GLP-1(7-37);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(lithocholoyl))-GLP-1(7-37);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-38);Lys²⁶(N^(ε)-(lithocholoyl))Arg³⁴-GLP-1(7-38);Gly⁸Lys²⁶(N^(ε)-(lithocholoyl))Arg³⁴-GLP-1(7-38);Arg^(26,34)Lys³⁶(N^(ε)-(lithocholoyl))-GLP-1(7-38);Arg^(26,34)Lys³⁸(N^(ε)-(lithocholoyl))-GLP-1(7-38);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(lithocholoyl))-GLP-1(7-38);Gly⁸Arg²⁶Lys³⁴(N^(ε)-(lithocholoyl))-GLP-1(7-39);Lys²⁶(N^(ε)-(lithocholoyl))Arg³⁴-GLP-1(7-39);Gly⁸Lys²⁶(N^(ε)-(lithocholoyl))Arg³⁴-GLP-1(7-39);Arg^(26,34)Lys³⁶(N^(ε)-(lithocholoyl))-GLP-1(7-39);Gly⁸Arg^(26,34)Lys³⁶(N^(ε)-(lithocholoyl))-GLP-1(7-39);Gly⁸Arg²⁶Lys³⁶(N^(ε)-(lithocholoyl))-GLP-1(7-40);Lys²⁶(N^(ε)-(lithocholoyl))Arg³⁴-GLP-1(7-40);Gly⁸Lys²⁶(N^(ε)-(lithocholoyl))Arg³⁴-GLP-1(7-40);Arg^(26,34)Lys³⁶(N^(ε)-(lithocholoyl))-GLP-1(7-40) andGly⁸Arg^(26,34)Lys³⁶(N^(ε)-(lithocholoyl))-GLP-1(7-40). Each of theseGLP-1 peptides constitutes an alternative embodiment of the presentinvention.

[0048] In a further embodiment of the present invention the GLP-1related impurities to be removed are selected from but not limited totruncated forms, all kinds of extended forms (extra amino acids, variousderivatives including esters etc.), deamidated forms, incorrectly foldedforms, forms with undesired glycosylation including sialylation. As anexample illustrating the present invention, histidine has a predominantpositive net charge below pH˜6.5, thus for cation exchange the pHelution gradient with a solvent comprising an organic solvent ormodifier could begin below pH 6.5 to remove a truncated form missinghistidine and end the gradient above pH 6.5 thereby subsequently elutingthe target GLP-1 moiety. As an alternative, the elution separating thetruncated form from the target GLP-1 moiety employing organic modifiercould be performed below pH 6.5 simply by a salt component (gradient orisocratically) at eluting conditions. As another alternative, theelution separating the truncated form from the target GLP-1 moietyemploying organic modifier could be performed below pH 6.5 by a gradientin the organic modifier from a lower to a higher content. As a secondexample, the carboxyl group of the C-terminal amino acid has apredominant negative net charge above pH-3.1, thus for anion exchangethe pH elution gradient with a solvent comprising an organic modifiercould begin above pH 3.1 to remove a form extended to an amide and endthe gradient below pH 3.1 thereby subsequently eluting the target GLP-1moiety. Alternatively, the elution separating the amide form from thetarget GLP-1 moiety employing organic modifier could be performed abovepH 3.1 simply by a salt component (gradient or isocratically) at elutingconditions. As a third example, aspartic acid has a predominant negativenet charge above pH˜4.4, thus for anion exchange the pH elution gradientwith a solvent comprising an organic modifier could begin above pH 4.4to remove a truncated form missing aspartic acid and end the gradientbelow pH 4.4 thereby subsequently eluting the target GLP-1 moiety.Alternatively, the elution separating the truncated form from the targetGLP-1 moiety employing organic modifier could be performed above pH 4.4simply by a salt component (gradient or isocratically) at elutingconditions. As a fourth example, glutamic acid has a predominantnegative net charge above pH˜4.4, thus for anion exchange the pH elutiongradient with a solvent comprising an organic modifier could begin abovepH 4.4 to elute the target GLP-1 moiety and end the gradient below pH4.4 thereby subsequently removing an extended form comprising an extraglutamic acid residue. Alternatively, the elution separating theextended form from the target GLP-1 moiety employing organic modifiercould be performed above pH 4.4 simply by a salt component (gradient orisocratically) at eluting conditions. As a fifth example, the aminogroup of the N-terminal amino acid has a predominant positive net chargebelow pH˜8.0, thus for cation exchange the pH elution gradient with asolvent comprising an organic modifier could begin below pH 8.0 toremove a form extended with an undesired acyl group and end the gradientabove pH 8.0 thereby subsequently eluting the target GLP-1 moiety.Alternatively, the elution separating the acylated form from the targetGLP-1 moiety employing organic modifier could be performed below pH 8.0simply by a salt component (gradient or isocratically) at elutingconditions. As a sixth example, the amino group of the N-terminal aminoacid has a predominant positive net charge below pH˜8.0, thus for cationexchange the pH elution gradient with a solvent comprising an organicmodifier could begin below pH 8.0 to elute the target GLP-1 moiety whichis extended with a desired acyl group and end the gradient above pH 8.0thereby subsequently removing the undesired non-extended form.Alternatively, the elution separating the acylated target GLP-1 moietyfrom the non-acylated form employing organic modifier could be performedbelow pH 8.0 simply by a salt component (gradient or isocratically) ateluting conditions. As a seventh example, tyrosine has a predominantnegative net charge above pH˜10.0, thus for anion exchange the pHelution gradient with a solvent comprising an organic modifier couldbegin above pH 10.0 to remove a truncated form missing a tyrosineresidue and end the gradient below pH 10.0 thereby subsequently elutingthe target GLP-1 moiety. Alternatively, the elution separating thetruncated form from the target GLP-1 moiety employing organic modifiercould be performed above pH 10.0 simply by a salt component (gradient orisocratically) at eluting conditions. As an eighth example, lysine has apredominant positive net charge below pH˜10.0, thus for cation exchangethe pH elution gradient with a solvent comprising an organic modifiercould begin below pH 10.0 to elute a target GLP-1 moiety acylated in theside chain of the lysine residue and end the gradient above pH 10.0thereby subsequently removing an undesired non-acylated form.Alternatively, the elution separating the acylated target GLP-1 moietyfrom the non-acylated form employing organic modifier could be performedbelow pH 10.0 simply by a salt component (gradient or isocratically) ateluting conditions. As a ninth example, arginine has a predominantpositive net charge below pH˜12.0, thus for anion exchange the pHelution gradient with a solvent comprising an organic modifier couldbegin below pH 12.0 to elute the target GLP-1 moiety and end thegradient above pH 12.0 thereby subsequently removing an undesired formcomprising an extra arginine residue. Alternatively, the elutionseparating the target GLP-1 moiety from the form comprising an extraarginine residue employing organic modifier could be performed below pH12.0 simply by a salt component (gradient or isocratically) at elutingconditions. (pK_(A)-values used in these examples are from: L. Stryer.Biochemistry, 3^(rd) edition, W. H. Freeman and Company, New York, Table2-1 page 21).

[0049] In a further embodiment of the present invention the impuritiesto be removed are not GLP-1 related.

[0050] Specific GLP-1 peptide examples of the above-mentioned method areseparation of Arg³⁴GLP-1 (7-37) and Arg³⁴GLP-1 (9-37) by cation exchangechromatography, GLP-1 (7-37) and GLP-1 (7-36) amide by anion exchangechromatography, GLP-1 (15-37) and GLP-1 (16-37) by anion exchangechromatography, Arg³⁴GLP-1 (7-37) and Arg³⁴Lys²⁶(N^(ε)-Glu)GLP -1 (7-37)by anion exchange chromatography,Arg³⁴Lys²⁶(N^(ε)-(γ-Glu-(N^(α)-tetradecanoyl)))GLP-1 (7-37) andArg³⁴Lys²⁶(N^(ε)-(γ-Glu-(N^(α)-tetradecanoyl)))Gly³⁷(N^(α)-(γ-Glu-(N^(α)-tetradecanoyl)))GLP-1(7-37) by cation exchange chromatography,Gly³⁷(N^(α)-(γ-Glu-(N^(α)-tetradecanoyl)))GLP-1 (7-37) and GLP-1 (7-37)by cation exchange chromatography, GLP-1 (19-37) and GLP-1 (20-37) byanion exchange chromatography,Arg³⁴Lys²⁶(N^(ε)-(γ-Glu-(N^(α)-tetradecanoyl)))GLP-1 (7-37) andArg³⁴GLP-1 (7-37) by cation exchange chromatography, and GLP-1 (7-37)and Arg³⁴GLP-1 (7-37) by cation exchange chromatography employing saltand/or pH gradients.

[0051] In a further embodiment of the present invention the peptide tobe purified is a GLP-2 peptide.

[0052] In a further embodiment of the present invention the peptide tobe purified is selected from GLP-2 (1-34), GLP-2 (1-33) as well asanalogues and derivatives thereof, in particular but not limited tohuman glucagon-like peptide-2 (hGLP-2), GLP-2(1-30); GLP-2(1-31);GLP-2(1-32); GLP-2(1-33); GLP-2(1-34), GLP-2(1-35), Lys²⁰GLP-2(1-33),Lys²⁰Arg³⁰GLP-2(1-33), Arg³⁰Lys³⁴GLP-2(1-34),Arg^(30,35)Lys³⁵GLP-2(1-35), Arg^(30,35)Lys²⁰GLP-2(1-35),Arg³⁵GLP-2(1-35), Lys²⁰(N^(ε)-tetradecanoyl)GLP-2(1-33);Lys^(20,30)-bis(N^(ε)-tetradecanoyl)GLP-2(1-33)Lys²⁰(N^(ε)-tetradecanoyl)Arg³⁰GLP-2(1-33);Arg³⁰Lys³⁵(N^(ε)-tetradecanoyl)GLP-2(1-35);Arg^(30,35)Lys²⁰(N^(ε)-tetradecanoyl)GLP-2(1-35);Arg³⁵Lys³⁰(N^(ε)-tetradecanoyl)GLP-2(1-35);Arg³⁰Lys³⁴(N^(ε)-tetradecanoyl)GLP-2(1-34);Lys²⁰(N^(ε)-(ω-carboxynonadecanoyl))GLP-2(1-33);Lys^(20,30)-bis(N^(ε)-(ω-carboxynonadecanoyl))GLP-2(1-33);Lys²⁰(N^(ε)-(ω-carboxynonadecanoyl))Arg³⁰GLP-2(1-33);Arg³⁰Lys³⁵(N^(ε)-(ω-carboxynonadecanoyl))GLP-2(1-35);Lys³⁰(N^(ε)-(γ-glutamyl(N^(α)-tetradecanoyl)))hGLP-2,Arg^(30,35)Lys²⁰(N^(ε)-(ω-carboxynonadecanoyl))GLP-2(1-35);Arg³⁵Lys³⁰(N^(ε)-(ω-carboxynonadecanoyl))GLP-2(1 -35); andArg³⁰Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))GLP-2(1-34).

[0053] The peptides or GLP-1 peptides can be produced by a method whichcomprises culturing or fermenting a host cell containing a DNA sequenceencoding the peptide or GLP-1 peptide and capable of expressing saidpeptide in a suitable nutrient medium under conditions permitting theexpression of the peptide, after which the resulting peptide or GLP-1peptide is recovered from the culture or fermentation broth.Hereinafter, culturing will be used to cover both culturing andfermenting and the like.

[0054] The medium used to culture the cells may be any conventionalmedium suitable for growing the host cells, such as minimal or complexmedia containing appropriate supplements. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedrecipes (e.g. in catalogues of the American Type Culture Collection).The peptide or GLP-1 peptide produced by the cells may then be recoveredfrom the culture medium by conventional procedures including, optionallylysis of cells, separating the host cells from the medium bycentrifugation or filtration, precipitating the proteinaceous componentsof the supernatant or filtrate by means of a salt, e.g. ammoniumsulphate, purification by conventional purification techniques, such aschromatographic techniques, if necessary, purification by ion exchangechromatography according to the present invention, and subsequently,subjecting to analytical tests, e.g. PAGE, IEF, if necessary, subjectingto further purification, if necessary, and isolation of the pure peptideor GLP-1 peptide.

[0055] During the recovery of the resulting peptide or GLP-1 peptidefrom the culture medium, but before purification by ion exchangechromatography according to the present invention, the mixturecomprising the peptide or GLP-1 peptide and related impurities mayoptionally be chemically modified by conventional techniques, e.g. byalkylation, acylation, ester formation or amide formation or the like.

[0056] The DNA sequence encoding the parent peptide or GLP-1 peptide maysuitably be of genomic or cDNA origin, for instance obtained bypreparing a genomic or cDNA library and screening for DNA sequencescoding for all or part of the peptide or GLP-1 peptide by hybridisationusing synthetic oligonucleotide probes in accordance with standardtechniques (see, for example, Sambrook, J, Fritsch, E F and Maniatis, T,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, New York, 1989). The DNA sequence encoding the peptide or GLP-1peptide may also be prepared synthetically by established standardmethods, e.g. the phosphoamidite method described by Beaucage andCaruthers, Tetrahedron Letters 22 (1981), 1859-1869, or the methoddescribed by Matthes et al., EMBO Journal 3 (1984), 801-805. The DNAsequence may also be prepared by polymerase chain reaction usingspecific primers, for instance as described in U.S. Pat. No. 4,683,202or Saiki et al., Science 239 (1988), 487-491.

[0057] The DNA sequence may be inserted into any vector which mayconveniently be subjected to recombinant DNA procedures, and the choiceof vector will often depend on the host cell into which it is to beintroduced. Thus, the vector may be an autonomously replicating vector,i.e. a vector which exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g. aplasmid. Alternatively, the vector may be one which, when introducedinto a host cell, is integrated into the host cell genome and replicatedtogether with the chromosome(s) into which it has been integrated.

[0058] The vector is preferably an expression vector in which the DNAsequence encoding the peptide or GLP-1 peptide is operably linked toadditional segments required for transcription of the DNA, such as apromoter. The promoter may be any DNA sequence which showstranscriptional activity in the host cell of choice and may be derivedfrom genes encoding proteins either homologous or heterologous to thehost cell. Examples of suitable promoters for directing thetranscription of the DNA encoding the peptide or GLP-1 peptide of theinvention in a variety of host cells are well known in the art, cf. forinstance Sambrook et al., supra.

[0059] The DNA sequence encoding the peptide or GLP-1 peptide may also,if necessary, be operably connected to a suitable terminator,polyadenylation signals, transcriptional enhancer sequences, andtranslational enhancer sequences. The recombinant vector of theinvention may further comprise a DNA sequence enabling the vector toreplicate in the host cell in question.

[0060] The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell or one whichconfers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin,chloramphenicol, neomycin, hygromycin or methotrexate.

[0061] To direct a peptide or GLP-1 peptide into the secretory pathwayof the host cells, a secretory signal sequence (also known as a leadersequence, prepro sequence or pre sequence) may be provided in therecombinant vector. The secretory signal sequence is joined to the DNAsequence encoding the GLP-1 peptide in the correct reading frame.Secretory signal sequences are commonly positioned 5′ to the DNAsequence encoding the peptide or GLP-1 peptide. The secretory signalsequence may be that normally associated with the peptide or GLP-1peptide or may be from a gene encoding another secreted protein.

[0062] The procedures used to ligate the DNA sequences coding for thepeptide or GLP-1 peptide, the promoter and optionally the terminatorand/or secretory signal sequence, respectively, and to insert them intosuitable vectors containing the information necessary for replication,are well known to persons skilled in the art (cf., for instance,Sambrook et al., supra).

[0063] The host cell into which the DNA sequence or the recombinantvector is introduced may be any cell which is capable of producing thepresent peptide or GLP-1 peptide and includes bacteria, vira, e.g.baculo virus, yeast, fungi, insect cells and higher eukaryotic cells.Examples of suitable host cells well known and used in the art are,without limitation, E. coli, Saccharomyces cerevisiae, or mammalian BHKor CHO cell lines.

[0064] Some of the peptides or GLP-1 peptides, can be produced accordingto conventional organic peptide synthetic chemistry. The resultingsynthetic mixture may then be chemically modified, e.g. by alkylation,acylation, ester formation or amide formation or the like, and purified,or purified as it is and then modified chemically as mentioned above.

[0065] Preparation of Factor VIIa

[0066] Human purified factor VIIa suitable for use in the presentinvention is preferably made by DNA recombinant technology, e.g. asdescribed by Hagen et al., Proc.Natl.Acad.Sci. USA 83: 2.412-2416, 1986or as described in European Patent No. 200.421 (ZymoGenetics). FactorVIIa produced by recombinant technology may be authentic factor VIIa ora more or less modified factor VIIa provided that such factor VIIa hassubstantially the same biological activity for blood coagulation asauthentic factor VIIa. Such modified factor VIIa may be produced bymodifying the nucleic acid sequence encoding factor VII either byaltering the amino acid codons or by removal of some of the amino acidcodons in the nucleic acid encoding the natural FVII by known means,e.g. by site-specific mutagenesis.

[0067] Factor VII may also be produced by the methods described by Brozeand Majerus, J.Biol.Chem. 255 (4): 1242-1247, 1980 and Hedner andKisiel, J.Clin.Invest. 71: 1836-1841, 1983. These methods yield factorVII without detectable amounts of other blood coagulation factors. Aneven further purified factor VII preparation may be obtained byincluding an additional gel filtration as the final purification step.Factor VII is then converted into activated FVIIa by known means, e.g.by several different plasma proteins, such as factor XIIa, IXa or Xa.Alternatively, as described by Bjoern et al. (Research Disclosure, 269September 1986, pp. 564-565), factor VII may be activated by passing itthrough an ion-exchange chromatography column, such as Mono Q®(Pharmacia fine Chemicals) or the like.

[0068] Modified or Inactivated FVIIa (FVIIai) may be Produced by theFollowing Methods

[0069] International Application No. WO 92115686 relates to modifiedFactor VIIa, polynucleic acid and mammalian cell lines for theproduction of modified Factor VIIa, and compositions comprising modifiedFactor VIIa for inhibiting blood coagulation.

[0070] Modified Factor VII may be encoded by a polynucleotide moleculecomprising two operatively linked sequence coding regions encoding,respectively, a pre-pro peptide and a gla domain of a vitaminK-dependent plasma protein, and a gla domain-less Factor VII protein,wherein upon expression said polynucleotide encodes a modified FactorVII molecule which does not significantly activate plasma Factors X orIX, and is capable of binding tissue factor.

[0071] The catalytic activity of Factor VIIa can be inhibited bychemical derivatization of the catalytic centre, or triad.Derivatization may be accomplished by reacting Factor VII with anirreversible inhibitor such as an organophosphor compound, a sulfonylfluoride, a peptide halomethyl ketone or an azapeptide, or by acylation,for example. Preferred peptide halomethyl ketones include PPACK(D-Phe-Pro-Arg chloromethyl-ketone; (see U.S. Pat. No. 4,318,904,incorporated herein by reference), D-Phe-Phe-Arg and Phe-Phe-Argchloromethylketone (FFR-cmk); and DEGRck (dansyl-Glu-Gly-Argchloromethylketone).

[0072] The catalytic activity of Factor VIIa can also be inhibited bysubstituting, inserting or deleting amino acids. In preferredembodiments amino acid substitutions are made in the amino acid sequenceof the Factor VII catalytic triad, defined herein as the regions, whichcontain the amino acids, which contribute to the Factor VIIa catalyticsite. The substitutions, insertions or deletions in the catalytic triadare generally at or adjacent to the amino acids which form the catalyticsite. In the human and bovine Factor VII proteins, the amino acids,which form a catalytic “triad”, are Ser₃₄₄, Asp₂₄₂, and His₁₉₃(subscript numbering indicating position in the sequence). The catalyticsites in Factor VII from other mammalian species may be determined usingpresently available techniques including, among others, proteinisolation and amino acid sequence analysis. Catalytic sites may also bedetermined by aligning a sequence with the sequence of other serineproteases, particularly chymotrypsin, whose active site has beenpreviously determined (Sigler et al., J. Mol. Biol., 35:143-164 (1968),incorporated herein by reference), and there from determining from saidalignment the analogous active site residues.

[0073] In preferred embodiments of human and bovine Factor VII, theactive site residue Ser₃₄₄ is modified, replaced with Gly, Met, Thr, ormore preferably, Ala. Such substitution could be made separately or incombination with substitution(s) at other sites in the catalytic triad,which includes His₁₉₃ and Asp₂₄₂.

[0074] The amino acids, which form the catalytic site in Factor VII,such as Ser₃₄₄, Asp₂₄₂, and His₁₉₃ in human and bovine Factor VII, mayeither be substituted or deleted. Within the present invention, it ispreferred to change only a single amino acid, thus minimizing thelikelihood of increasing the antigenicity of the molecule or inhibitingits ability to bind tissue factor, however two or more amino acidchanges (substitutions, additions or deletions) may be made andcombinations of substitution(s), addition(s) and deletion(s) may also bemade. In a preferred embodiment for human and bovine Factor VII, Ser₃₄₄is preferably substituted with Ala, but Gly, Met, Thr or other aminoacids can be substituted. It is preferred to replace Asp with Glu and toreplace His with Lys or Arg. In general, substitutions are chosen todisrupt the tertiary protein structure as little as possible. The modelof Dayhoff et al. (in Atlas of Protein Structure 1978, Nat'l Biomed.Res. Found., Washington, D.C.), incorporated herein by reference, may beused as a guide in selecting other amino acid substitutions. One mayintroduce residue alterations as described above in the catalytic siteof appropriate Factor VII sequence of human, bovine or other species andtest the resulting protein for a desired level of inhibition ofcatalytic activity and resulting anticoagulant activity as describedherein. For the modified Factor VII the catalytic activity will besubstantially inhibited, generally less than about 5% of the catalyticactivity of wild-type Factor VII of the corresponding species, morepreferably less than about 1%.

[0075] The modified Factor VII may be produced through the use ofrecombinant DNA techniques.

[0076] The amino acid sequence alterations may be accomplished by avariety of techniques. Modification of the DNA sequence may be bysite-specific mutagenesis. Techniques for site-specific mutagenesis arewell known in the art and are described by, for example, Zoller andSmith (DNA 3:479-488, 1984). Thus, using the nucleotide and amino acidsequences of Factor VII, one may introduce the alteration(s) of choice.The modified FVIIa may also be produced by chemical methods.

[0077] FFR-FVIIa (that is, D-Phe-Phe-Arg-FVIIa)

EXAMPLE FFR Chloromethyl Ketone

[0078] Blockage of the active site of FVIIa with FFR chloromethylketone.

[0079] Blockage of the active site serine and histidine withchloromethyl ketone is a well-known method for irreversible inactivationof serine proteases. In order to optimise the blockage of a givenprotease it is important to choose a chloromethyl ketone derivative,which reacts specifically with the active site and with a fast on-rate.Such derivatives can be developed by attachment to the chloromethylketone group of an oligopeptide, which interacts, with thesubstrate-binding pocket of the particular serine protease of interest.

[0080] Glutamyl-Glycyl-Arginine chloromethyl ketone (EGR-ck or itsDansyl derivative, DEGR-ck) (S. Higashi, H. Nishimura, S. Fujii, K.Takada, S. Iwanaga, (1992) J. Biol. Chem. 267, 17990) orProlyl-Phenyl-Arginine chloromethyl ketone (PFR-ck) (J. H. Lawson, S.Butenas, K. Mann, (1992) J. Biol. Chem. 267, 4834; J. Contrino, G. A.Hair, M. A. Schmeizl, F. R. Rickles, D. L. Kreutzer (1994) Am. J.Pathol. 145, 1315) have been applied as active site inhibitors of FVIIa.Compared with these chloromethyl ketones application of FFRck representsa rate increase of 10-70 fold.

[0081] The specificity of the reaction with FFR-chloromethyl ketonederivative of FVIIa was checked by HPLC and peptide mapping which showedthat FVIIa had reacted with FFR-chloromethyl ketone in a 1:1 ratio suchthat >98% could be recovered as the expected product derivatized athistidine 193.

[0082] Inactivation of FVIIa by Various Chloromethyl Ketones

[0083] 3 μM FVIIa was incubated with 12 μM of chloromethyl ketonederivative in 50 mM TrisHCl, 100 mM NaCl, 5 mM CaCl₂, 0.01% Tween-80, pH7.4. Samples were withdrawn at various time intervals as indicated andand diluted 20 times for activity measurements in 50 mM TrisHCl, 100 mMNaCl, 5 mM CaCl₂, 0.01% Tween-80, pH 7.4 containing 1 mMIle-Pro-Arg-pNA. The residual FVIIa activity was measured by theincrease in absorbance at 405 nm.

[0084] Usually, the mixture comprising the peptide or GLP-1 peptide andrelated impurities to be purified by ion exchange chromatographyaccording to the present invention, will also contain amino acids, smallpeptides, large peptides, unrelated proteins, reactants, cell debris,HCP, endotoxins, and/or vira depending on whether recombinant DNAtechniques and/or chemical modification techniques have been used orwhether organic peptide synthetic chemistry techniques have been used.

[0085] Thus, any method, such as an industrial method, for producing apure peptide or GLP-1 peptide, which includes an IFC process accordingto the present invention is also an aspect of the present application.

[0086] Accordingly, the present invention relates in a further aspect toan industrial method for producing a pure peptide or GLP-1 peptide, themethod including a cation exchange chromatography process for purifyinga peptide from a mixture comprising said peptide or GLP-1 peptide andrelated impurities, comprising the steps of:

[0087] separating said peptide or GLP-1 peptide and said relatedimpurities of said mixture by elution in a solution comprising anorganic modifier, water, optionally a salt component, and optionally abuffer, with a linear or step gradient or isocratically in saltcomponent and/or with a linear or step pH-gradient or at a constantpH-value, wherein the pH-gradient or pH-value should be in the rangewhere said peptide or GLP-1 peptide has a positive local or overall netcharge different from the local or overall positive net charge of saidrelated impurities so as to remove said related impurities.

[0088] The present invention relates in a further aspect to a method forisolating a GLP-1 peptide, the method including purification of a GLP-1peptide from a mixture comprising said peptide and related impuritiesvia a cation exchange chromatography process, the cation exchangechromatography process comprising the step of:

[0089] separating said GLP-1 peptide and said related impurities of saidmixture by elution in a solution comprising an organic modifier, water,optionally a salt component, and optionally a buffer, with a linear orstep gradient or isocratically in salt component and/or with a linear orstep pH-gradient or at a constant pH-value, wherein the pH-gradient orpH-value should be in the range where said peptide has a positive localor overall net charge different from the local or overall positive netcharge of said related impurities so as to remove said relatedimpurities; and subsequently, if necessary, subjecting to analyticaltests and/or further purification, and isolating said peptide in aconventional manner.

[0090] The present invention relates in a further aspect to a method forisolating a GLP-1 peptide, the method including purification of a GLP-1peptide from a mixture comprising said peptide and related or unrelatedimpurities via a cation exchange chromatography process, the cationexchange chromatography process comprising the step of:

[0091] separating said GLP-1 peptide and said related impurities of saidmixture by elution in a solution consisting essentially of an organicmodifier, water, optionally a salt component, and optionally a buffer,with a linear or step gradient or isocratically in salt component and/orwith a linear or step pH-gradient or at a constant pH-value, wherein thepH-gradient or pH-value should be in the range where said peptide has apositive local or overall net charge different from the local or overallpositive net charge of said related impurities so as to remove saidrelated impurities; and subsequently, if necessary, subjecting toanalytical tests and/or further purification, and isolating said peptidein a conventional manner.

[0092] The present invention relates in a further aspect to anindustrial method for producing a pure peptide or GLP-1 peptide, themethod including an anion exchange chromatography process for purifyinga peptide from a mixture comprising said peptide or GLP-1 peptide andrelated impurities, comprising the steps of:

[0093] separating said peptide or GLP-1 peptide and said relatedimpurities of said mixture by elution in a solution comprising anorganic modifier, water, optionally a salt component, and optionally abuffer, with a linear or step gradient or isocratically in saltcomponent and/or with a linear or step pH-gradient or at a constantpH-value, wherein the pH-gradient or pH-value should be in the rangewhere said peptide or GLP-1 peptide has a negative local or overall netcharge different from the local or overall negative net charge of saidrelated impurities so as to remove said related impurities.

[0094] The present invention relates in a still further aspect to amethod for isolating a GLP-1 peptide, the method including purificationof a GLP-1 peptide from a mixture comprising said peptide and relatedimpurities via an anion exchange chromatography process, the anionexchange chromatography process comprising the step of:

[0095] separating said GLP-1 peptide and said related impurities of saidmixture by elution in a solution comprising an organic modifier, water,optionally a salt component, and optionally a buffer, with a linear orstep gradient or isocratically in salt component and/or with a linear orstep pH-gradient or at a constant pH-value, wherein the pH-gradient orpH-value should be in the range where said peptide has a negative localor overall net charge different from the local or overall negative netcharge of said related impurities so as to remove said relatedimpurities; and subsequently, if necessary, subjecting to analyticaltests and/or further purification, and isolating said peptide in aconventional manner.

[0096] The present invention relates in a still further aspect to amethod for isolating a GLP-1 peptide, the method including purificationof a GLP-1 peptide from a mixture comprising said peptide and related orunrelated impurities via an anion exchange chromatography process, theanion exchange chromatography process comprising the step of:

[0097] separating said GLP-1 peptide and said related impurities of saidmixture by elution in a solution consisting essentially of an organicmodifier, water, optionally a salt component, and optionally a buffer,with a linear or step gradient or isocratically in salt component and/orwith a linear or step pH-gradient or at a constant pH-value, wherein thepH-gradient or pH-value should be in the range where said peptide has anegative local or overall net charge different from the local or overallnegative net charge of said related impurities so as to remove saidrelated impurities; and subsequently, if necessary, subjecting toanalytical tests and/or further purification, and isolating said peptidein a conventional manner.

[0098] Any possible combination of two or more of the embodimentsdescribed herein, is comprised within the scope of the presentinvention.

[0099] The term “an organic modifier”, as used herein, is intended toinclude an organic solvent or organic compound soluble in water ormixtures thereof, which modifier induces a favorable and changedselectivity between the unwanted related impurity or impurities and theGLP-1 peptide and the ion exchanger. Whether or not a selected modifierinduces said selectivity will usually depend on the related impurity orimpurities, and may be tested by trial-and-error. The organic modifierincludes but is not limited to C₁₋₆-alkanol, C₁₋₆-alkenol orC₁₋₆-alkynol, urea, guanidine.HCl, or C₁₋₆-alkanoic acid, such as aceticacid, C₂₋₆-glycol, C₃₋₇-polyalcohol including sugars, or mixturesthereof.

[0100] The term “C₁₋₆-alkanol”, “C₁₋₆-alkenol” or “C₁₋₆-alkynol”, asused herein, alone or in combination is intended to include thoseC₁₋₆-alkane, C₁₋₆-alkene or C₁₋₆-alkyne groups of the designated lengthin either a linear or branched or cyclic configuration whereto is linkeda hydroxyl (—OH) (cf. Morrison & Boyd, Organic Chemistry, 4^(th) ed).Examples of linear alcohols are methanol, ethanol, n-propanol, allylalcohol, n-butanol, n-pentanol and n-hexanol. Examples of branchedalcohols are 2-propanol and tert-butyl alcohol. Examples of cyclicalcohols are cyclo propyl alcohol and 2-cyclohexen-1-ol.

[0101] The term “C₁₋₆-alkanoic acid”, as used herein, is intended toinclude a group of the formula R'COOH wherein R′ is H or C₁₋₅alkyl, suchas acetic, propionic, butyric, methylbutyric, or valeric acid (cf.Morrison & Boyd, Organic Chemistry, 4^(th) ed).

[0102] The term “C₁₋₅-alkyl”, as used herein, is intended to include abranched or straight alkyl group having from one to five carbon atoms.Typical C₁₋₅-alkyl groups include, but are not limited to, methyl,ethyl, n-propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl,pentyl, iso-pentyl, and the like (cf. Morrison & Boyd, OrganicChemistry, 4^(th) ed).

[0103] The term “C₂₋₆-glycol”, as used herein, is intended to include aC₂₋₆-alkane containing two hydroxyl groups on different carbon atomswhich may be adjacent or not. A typical C₂₋₆-glycol include, but is notlimited to 1,2-ethanediol, 1,2-propanediol, or 2-methyl-2,4-pentanediol(cf. Morrison & Boyd, Organic Chemistry, 4^(th) ed).

[0104] The term “C₂₋₆-alkane”, as used herein, is intended to include abranched or straight alkane group having from two to six carbon atoms.Typical C₂₋₆-alkane groups include, but are not limited to ethane,propane, iso-propane, butane, iso-butane, pentane, hexane and the like(cf. Morrison & Boyd, Organic Chemistry, 4^(th) ed).

[0105] The term “C₃₋₇-polyalcohol including sugars”, as used herein, isintended to include a group of the formula HOCH₂(CHOH)_(n)CH₂OH whereinn is an integer from 1-5, and monosaccharides such as mannose, glucose(cf. Morrison & Boyd, Organic Chemistry, 4^(th) ed).

[0106] The term “GLP-1 peptide”, as used herein, is intended todesignate GLP-1 (7-37), GLP-1 (7-36) amide as well as analogues andderivatives thereof, which are capable of being produced by conventionalrecombinant DNA techniques as well as conventional synthetic methods.Such GLP-1 peptides include but are not limited to native glucagon-likepeptide-1, for instance such peptide fragments which comprises GLP-1(7-37) and functional derivatives thereof as disclosed in WO 87/06941;such peptide fragments which comprise GLP-1 (7-36) and functionalderivatives thereof as disclosed in WO 90/11296; such analogues of theactive GLP-1 peptides 7-34, 7-35, 7-36, and 7-37 as disclosed in WO91/11457; such GLP-1 derivatives in which a lipophilic substituent isattached to at least one amino acid residue as disclosed in WO 98108871;such N-terminal truncated fragments of GLP-1 as disclosed in EP0699686-A2; and such GLP-1 analogues and derivatives that include anN-terminal imidazole group as disclosed in EP 0708179-A2.

[0107] The term “GLP-2 peptide”, as used herein, is intended todesignate GLP-2 (1-35), GLP-2 (1-34), GLP-2 (1-33) as well as analoguesand derivatives thereof, which are capable of being produced byconventional recombinant DNA techniques as well as conventionalsynthetic methods. Such GLP-2 peptides include but are not limited tonative glucagon-like peptide-2, GLP-2 derivatives in which a lipophilicsubstituent is attached to at least one amino acid residue as disclosedin WO 98/08872, human glucagon-like peptide-2 (hGLP-2), GLP-2(1-30);GLP-2(1-31); GLP-2(1-32); GLP-2(1-33); GLP-2(1-34), GLP-2(1-35),Lys²⁰GLP-2(1-33), Lys²⁰Arg³⁰GLP-2(1-33), Arg³⁰Lys³⁴GLP-2(1-34),Arg³⁰Lys³⁵GLP-2(1-35), Arg^(30,35)Lys²⁰GLP-2(1-35), Arg³⁵GLP-2(1-35),Lys²⁰(N^(ε)-tetradecanoyl)GLP-2(1-33);Lys^(20,30)-bis(N^(ε)-tetradecanoyl)GLP-2(1-33);Lys²⁰(N^(ε)-tetradecanoyl)Arg³⁰GLP-2(1-33);Arg³⁰Lys³⁵(N^(ε)-tetradecanoyl)GLP-2(1-35);Arg^(30,35)Lys²⁰(N^(ε)-tetradecanoyl)GLP-2(1-35);Arg³⁵Lys³⁰(N^(ε)-tetradecanoyl)GLP-2(1-35);Arg³⁰Lys³⁴(N^(ε)-tetradecanoyl)GLP-2(1-34);Lys²⁰(N^(ε)-(ω-carboxynonadecanoyl))GLP-2(1-33);Lys^(20,30)-bis(N^(ε)-(ω-carboxynonadecanoyl))GLP-2(1-33);Lys²⁰(N^(ε)-(ω-carboxynonadecanoyl))Arg³⁰GLP-2(1-33);Arg³⁰Lys³⁵(N^(ε)-(ω-carboxynonadecanoyl))GLP-2(1-35);Lys³⁰(N^(ε)-(γ-glutamyl(N^(α)-tetradecanoyl)))hGLP-2,Lys³⁰(N^(ε)-(γ-glutamyl(N^(α)-hexadecanoyl)))hGLP-2,Arg^(30,35)Lys²⁰(N^(ε)-(ω-carboxynonadecanoyl))GLP-2(1-35);Arg³⁵Lys³⁰(N^(ε)-(ω-carboxynonadecanoyl))GLP-2(1-35); andArg³⁰Lys³⁴(N^(ε)-(ω-carboxynonadecanoyl))GLP-2(1-34).

[0108] The term “analogues” as used herein, is intended to designate apeptide wherein one or more amino acid residues of the parent peptidehave been substituted by another amino acid residue and/or wherein oneor more amino acid residues of the parent peptide have been deletedand/or wherein one or more amino acid residues have been added to theparent peptide. Such addition can take place either at the N-terminalend or at the C-terminal end of the parent peptide or both.

[0109] The term “derivatives” as used herein, is intended to designate apeptide in which one or more of the amino acid residues of the parentpeptide have been chemically modified, e.g. by alkylation, acylation,ester formation or amide formation or the like.

[0110] The term “salt component” as used herein, is intended to includeany organic or inorganic salt, including but not limited to NaCl, KCl,NH₄Cl, CaCl₂, sodium acetate, potassium acetate, ammonium acetate,sodium citrate, potassium citrate, ammonium citrate, sodium sulphate,potassium sulphate, ammonium sulphate, calcium acetate or mixturesthereof (cf. Remington's Pharmaceutical Sciences, Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, or Remington: The Science andPractice of Pharmacy, 19th Edition (1995), or handbooks fromAmersham-Pharmacia Biotech).

[0111] The term “a buffer” as used herein, is intended to include anybuffer including but not limited to: citrate buffers, phosphate buffers,tris buffers, borate buffers, lactate buffers, glycyl glycin buffers,arginine buffers, carbonate buffers, acetate buffers, glutamate buffers,ammonium buffers, glycin buffers, alkylamine buffers, aminoethyl alcoholbuffers, ethylenediamine buffers, tri-ethanol amine, imidazole buffers,pyridine buffers and barbiturate buffers and mixtures thereof (cf.Remington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co.,Easton, Pa., 1990, or Remington: The Science and Practice of Pharmacy,19th Edition (1995), or handbooks from Amersham-Pharmacia Biotech).

[0112] The choice of starting pH, buffer and ionic strength is doneaccording to well-known techniques such as conventional test-tubemethods, cf. e.g. handbooks from Amersham-Pharmacia Biotech. Thechromatographic ion exchange resin is chosen depending on the specificGLP-1 peptide to be purified and the conditions employed, such as pH,buffer, ionic strength etc., which are known to the person skilled inthe art (that is, typically, pH below the isoelectric point (pI) of theGLP-1 peptide for cation exchange resins and pH above pI of the GLP-1peptide for anion exchange resins, a sufficient buffer strength tomaintain the desired pH, and a sufficiently low ionic strength possiblyinduced by the salt concentration), and includes but is not limited toSepharose resins, Sephadex resins, Streamline resins, and Source resinsfrom Amersham-Pharmacia Biotech, HyperD resins, Trisacryl resins, andSpherosil resins from BioSepra, TSKgel resins and Toyopearl resins fromTosoHaas, Fractogel EMD resins from Merck, Poros resins from PerseptiveBiosystems, Macro-Prep resins from BioRAD, Express-ion resins fromWhatman etc.

[0113] The term “a solution consisting essentially of an organicmodifier, water, optionally a salt component and optionally a buffer” asused herein, is intended to mean a solution containing one or moreorganic modifiers, water, one or more salt components or no saltcomponent and one or more buffers or no buffer, and optionally one ormore further conventional components which the person skilled in the artwould consider adding, according to conventional ion exchangechromatography processes.

[0114] The term “related impurities” as used herein, is intended to meanone or more impurities with a different local or overall net charge fromthe GLP-1 peptide, for instance truncated forms, all kinds of extendedforms (extra amino acids, various derivatives including esters etc.),deamidated forms, incorrectly folded forms, forms with undesiredglycosylation including sialylation, and others. It follows then that“unrelated impurities” as used herein, is intended to cover impuritieswhich are different from related impurities.

[0115] The term “a constant pH-value”, as used herein is intended tomean that the pH-value may be constant, such as in the presence of abuffer or may vary typically within 3 pH units, if no buffer is present.

[0116] The term “with a linear or step pH-gradient”, as used herein isintended to mean that the pH-value changes during the elution from alower to a higher pH, or from a higher to a lower pH. Such change in pHis usually generated with a buffer and/or by addition of an inorganic ororganic acid or base, e.g. HCl, NaOH, H₂O, acetic acid, NH₃, KOH, H₂SO₄,citric acid. A pH gradient for cation exchange would usually be from alower to a higher pH, and for anion exchange from a higher to a lowerpH.

[0117] The term “with a linear or step gradient or isocratically in saltcomponent”, as used herein is intended to mean that the saltconcentration changes during the elution from a lower to a higherconcentration or is constant.

[0118] The present invention also relates to the following aspects:

[0119] Aspect 1. A cation exchange chromatography process for purifyinga GLP-1 peptide from a mixture comprising said peptide and related orunrelated impurities, comprising the step of:

[0120] separating said GLP-1 peptide and said related or unrelatedimpurities of said mixture by elution in a solution consistingessentially of an organic modifier, water, optionally a salt component,and optionally a buffer, with a linear or step gradient or isocraticallyin salt component and/or with a linear or step pH-gradient or at aconstant pH-value, wherein the pH-gradient or pH-value should be in therange where said peptide has a positive local or overall net chargedifferent from the local or overall positive net charge of said relatedor unrelated impurities so as to remove said related or unrelatedimpurities.

[0121] Aspect 2. A cation exchange chromatography process for purifyinga GLP-1 peptide from a mixture comprising said peptide and relatedimpurities, comprising the step of:

[0122] separating said GLP-1 peptide and said related impurities of saidmixture by elution in a solution consisting essentially of an organicmodifier, water, optionally a salt component, and optionally a buffer,with a linear or step gradient or isocratically in salt component and/orwith a linear or step pH-gradient or at a constant pH-value, wherein thepH-gradient or pH-value should be in the range where said peptide has apositive local or overall net charge different from the local or overallpositive net charge of said related impurities so as to remove saidrelated impurities.

[0123] Aspect 3. An anion exchange chromatography process for purifyinga GLP-1 peptide from a mixture comprising said peptide and related orunrelated impurities, comprising the step of:

[0124] separating said GLP-1 peptide and said related or unrelatedimpurities of said mixture by elution in a solution consistingessentially of an organic modifier, water, optionally a salt component,and optionally a buffer, with a linear or step gradient or isocraticallyin salt component and/or with a linear or step pH-gradient or at aconstant pH-value, wherein the pH-gradient or pH-value should be in therange where said peptide has a negative local or overall net chargedifferent from the local or overall negative net charge of said relatedor unrelated impurities so as to remove said related or unrelatedimpurities.

[0125] Aspect 4. An anion exchange chromatography process for purifyinga GLP-1 peptide from a mixture comprising said peptide and relatedimpurities, comprising the step of:

[0126] separating said GLP-1 peptide and said related impurities of saidmixture by elution in a solution consisting essentially of an organicmodifier, water, optionally a salt component, and optionally a buffer,with a linear or step gradient or isocratically in salt component and/orwith a linear or step pH-gradient or at a constant pH-value, wherein thepH-gradient or pH-value should be in the range where said peptide has anegative local or overall net charge different from the local or overallnegative net charge of said related impurities so as to remove saidrelated impurities.

[0127] Aspect 5. The process according to any one of aspects 1-4 whereinthe ratio of organic modifier to water on a weight percent basis is from1:99 to 99:1.

[0128] Aspect 6. The process according to any one of aspects 1-5 whereinsaid organic modifier is selected from C₁₋₆-alkanol, C₁₋₆-alkenol orC₁₋₆-alkynol, urea, guanidine, or C₁₋₆-alkanoic acid, C₂₋₆-glycol, orC₃₋₇-polyalcohol including sugars.

[0129] Aspect 7. The process according to any one of aspects 1-6 whereinsaid salt component is selected from any organic or inorganic salt,preferably NaCl, KCl, NH₄Cl, CaCl₂, sodium acetate, potassium acetate,ammonium acetate, sodium citrate, potassium citrate, ammonium citrate,sodium sulphate, potassium sulphate, ammonium sulphate, calcium acetateor mixtures thereof.

[0130] Aspect 8. The process according to any one of aspects 1-6 whereinno salt component is present.

[0131] Aspect 9. The process according to any one of aspects 1-7 whereinsaid gradient in salt component is a step or linear gradient in the saltcomponent.

[0132] Aspect 10. The process according to aspect 9 wherein said saltcomponent is present in a concentration selected from the range of 0.1mmol/kg to 3000 mmol/kg.

[0133] Aspect 11. The process according to any one of aspects 1-10wherein said buffer is independently selected from citrate buffers,phosphate buffers, tris buffers, borate buffers, lactate buffers, glycylglycin buffers, arginine buffers, carbonate buffers, acetate buffers,glutamate buffers, ammonium buffers, glycin buffers, alkylamine buffers,aminoethyl alcohol buffers, ethylenediamine buffers, tri-ethanol amine,imidazole buffers, pyridine buffers and barbiturate buffers and mixturesthereof.

[0134] Aspect 12. The process according to any one of aspects 1-11wherein said buffer is present in a concentration selected from therange of 0.1 mmol/kg to 500 mmol/kg.

[0135] Aspect 13. The process according to any one of aspects 1-10wherein no buffer is present.

[0136] Aspect 14. A method for isolating a peptide, the method includingpurification of a GLP-1 peptide from a mixture comprising said peptideand related impurities via a cation exchange chromatography process, thecation exchange chromatography process comprising the steps of:

[0137] separating said GLP-1 peptide and said related impurities of saidmixture by elution in a solution consisting essentially of an organicmodifier, water, optionally a salt component, and optionally a buffer,with a linear or step gradient or isocratically in salt component and/orwith a linear or step pH-gradient or at a constant pH-value, wherein thepH-gradient or pH-value should be in the range where said peptide has apositive local or overall net charge different from the local or overallpositive net charge of said related impurities so as to remove saidrelated impurities;

[0138] and subsequently, if necessary, subjecting to analytical testsand/or further purification, and isolating said peptide in aconventional manner.

[0139] Aspect 15. A method for isolating a peptide, the method includingpurification of a GLP-1 peptide from a mixture comprising said peptideand related impurities via an anion exchange chromatography process, theanion exchange chromatography process comprising the steps of:

[0140] separating said GLP-1 peptide and said related impurities of saidmixture by elution in a solution consisting essentially of an organicmodifier, water, optionally a salt component, and optionally a buffer,with a linear or step gradient or isocratically in salt component and/orwith a linear or step pH-gradient or at a constant pH-value, wherein thepH-gradient or pH-value should be in the range where said peptide has anegative local or overall net charge different from the local or overallnegative net charge of said related impurities so as to remove saidrelated impurities;

[0141] and subsequently, if necessary, subjecting to analytical testsand/or further purification, and isolating said peptide in aconventional manner.

[0142] Aspect 16. The process or method according to any one of aspects1-15 wherein said peptide to be purified is selected from GLP-1 (7-37),GLP-1 (7-36) amide as well as analogues and derivatives thereof.

EXAMPLES

[0143] The present invention is further illustrated by the followingexamples which, however, are not to be construed as limiting the scopeof protection. The features disclosed in the foregoing description andin the following examples may, both separately and in any combinationthereof, be material for realising the invention in diverse formsthereof.

Example 1

[0144] Arg³⁴GLP-1₍₇₋₃₇₎ was expressed in yeast (Sacch. cerevisiae) byconventional recombinant DNA technology e.g as described in WO 98/08871.Arg³⁴GLP-1₍₇₋₃₇₎ fermentation broth was purified by a conventionalcation exchange chromatography capture step. The pool containingArg³⁴GLP-1₍₇₋₃₇₎ and the truncated form, Arg³⁴GLP-1₍₉₋₃₇₎, as animpurity was adjusted to pH 3.1, and 1 ml of the resulting solution wasapplied to a 6.5 ml Ceramic S HyperD F (Bio-Sepra S. A.) columnequilibrated with 32.5 ml 1.54% (w/w) tri-sodium citrate, 0.6% (w/w)succinic acid, 1.09% (w/w) di-sodium hydrogen phosphate di-hydrate,pH˜3.2. The column was washed with 6.5 ml equilibration solution, andelution was performed with a linear pH gradient from 3.2 to 8.0 (1.54%(w/w) tri-sodium citrate, 0.6% (w/w) succinic acid, 1.09% (w/w)di-sodium hydrogen phosphate di-hydrate, pH 8.0) followed by 13 ml ofisocratic elution at pH 8.0. A chromatogram is shown in FIG. 1. Nodistinct peaks or separation between the truncated form and the targetGLP-1 moiety were obtained.

Example 2

[0145] Arg³⁴GLP-1₍₇₋₃₇₎ was expressed and captured by cation exchange asdescribed in Example 1. The pool containing Arg³⁴GLP-1₍₇₋₃₇₎ and thetruncated form, Arg³⁴GLP-1₍₉₋₃₇₎, as an impurity was adjusted to pH 3.1,and 1 ml of the resulting solution was applied to a 20 ml Source 30S(Amersham Pharmacia Biotech) column equilibrated with 100 ml 1.54% (w/w)tri-sodium citrate, 0.6% (w/w) succinic acid, 1.09% (w/w) di-sodiumhydrogen phosphate di-hydrate, pH˜3.2. The column was washed with 20 mlequilibration solution, and elution was performed with a linear pHgradient from 3.2 to 8.0 (1.54% (w/w) tri-sodium citrate, 0.6% (w/w)succinic acid, 1.09% (w/w) di-sodium hydrogen phosphate di-hydrate, pH8.0) followed by 40 ml of isocratic elution at pH 8.0. No distinct peaksor separation between the truncated form and the target GLP-1 moietywere obtained.

Example 3

[0146] Arg³⁴GLP-1₍₇₋₃₇₎ was expressed and captured by cation exchange asdescribed in Example 1. The pool containing Arg³⁴GLP-1₍₇₋₃₇₎ and thetruncated form, Arg³⁴GLP-1₍₉₋₃₇₎, as an impurity was adjusted to pH 3.1,and 1 ml of the resulting solution was applied to a 20 ml Source 30S(Amersham Pharmacia Biotech) column equilibrated with 100 ml 0.77% (w/w)tri-sodium citrate, 0.3% (w/w) succinic acid, 0.55% (w/w) di-sodiumhydrogen phosphate di-hydrate, pH˜3.2. The column was washed with 20 mlequilibration solution. Elution was performed with a linear pH gradientfrom 3.2 to 8.0 (0.77% (w/w) tri-sodium citrate, 0.3% (w/w) succinicacid, 0.55% (w/w) di-sodium hydrogen phosphate di-hydrate) followed by40 ml of isocratic elution at pH 8.0. Subsequent elution at pH 8.0 wasperformed with a linear salt gradient from 0.0 to 1.0 M NaCl (0.77%(w/w) tri-sodium citrate, 0.3% (w/w) succinic acid, 0.55% (w/w)di-sodium hydrogen phosphate di-hydrate, pH 8.0) followed by 40 ml ofisocratic elution at 1.0 M NaCl. No distinct peaks or separation betweenthe truncated form and the target GLP-1 moiety were obtained.

Example 4

[0147] Arg³⁴GLP-1₍₇₋₃₇₎ was expressed and captured by cation exchange asdescribed in Example 1. 2 volumes of water was added to the poolcontaining Arg³⁴GLP-1₍₇₋₃₇₎ and the truncated form, Arg³⁴GLP-1₍₉₋₃₇₎, asan impurity, and the solution was adjusted to pH 3.5. 25.5 ml of theresulting solution was applied to a 20 ml Source 30S (Amersham PharmaciaBiotech) column equilibrated with 100 ml 0.21% (w/w) tri-sodium citrate,0.08% (w/w) succinic acid, 0.15% (w/w) di-sodium hydrogen phosphatedi-hydrate, 45% (w/w) ethanol, pH˜3.2. The column was washed with 20 mlequilibration solution, and elution was performed with a linear pHgradient from 3.2 to 8.0 (0.21% (w/w) tri-sodium citrate, 0.08% (w/w)succinic acid, 0.15% (w/w) di-sodium hydrogen phosphate di-hydrate, 45%(w/w) ethanol, pH 8.0) followed by 40 ml of isocratic elution at pH 8.0.A chromatogram is shown in FIG. 2. Distinct peaks and separation betweenthe truncated form and the target GLP-1 moiety were obtained by additionof ethanol to the chromatographic solutions. Insignificant differencesin set up between this and Example 2 are: different batch, pH, andaqueous dilution of the sample for application, higher load, and lowerbuffer concentration.

Example 5

[0148] Arg³⁴GLP-1₍₇₋₃₇₎ was expressed and captured by cation exchange asdescribed in Example 1. The pool containing Arg³⁴GLP-1₍₇₋₃₇₎ and thetruncated form, Arg³⁴GLP-1₍₉₋₃₇₎, as an impurity was adjusted to pH 3.1,and 5 ml of the resulting solution was applied to a 20 ml Source 30S(Amersham Pharmacia Biotech) column equilibrated with 100 ml 0.85% (w/w)tri-sodium citrate, 0.33% (w/w) succinic acid, 0.6% (w/w) di-sodiumhydrogen phosphate di-hydrate, 45% (w/w) ethanol, pH˜3.2. The column waswashed with 20 ml equilibration solution, and elution was performed witha linear pH gradient from ˜3.2 to ˜5.0 (0.85% (w/w) tri-sodium citrate,0.33% (w/w) succinic acid, 0.6% (w/w) di-sodium hydrogen phosphatedi-hydrate, 45% (w/w) ethanol) followed by 60 ml of isocratic elution atpH 8.0 (0.85% (w/w) tri-sodium citrate, 0.33% (w/w) succinic acid, 0.6%(w/w) di-sodium hydrogen phosphate di-hydrate, 45% (w/w) ethanol, pH8.0). A chromatogram is shown in FIG. 3. Distinct peaks and separationbetween the truncated form and the target GLP-1 moiety were obtained byaddition of ethanol to the chromatographic solutions. RP-HPLC analysisfor identification/verification of collected peaks was carried out on aC₁₈-substituted 120 Å silica (YMC) 4.0×250 mm column with 5 μmparticles. Buffer A consisted of 0.15 M (NH₄)₂SO₄ in 7.8% (w/w)acetonitrile, pH 2.5, and buffer B contained 63.4% (w/w) acetonitrile.Linear gradients from 37-41% B in 12 min followed by 41-100% B in 15 minwere run at a flow rate of 1 ml/min. The chromatographic temperature waskept at 60° C. and UV detection was performed at 214 nm. Analyticalresults were: Arg³⁴GLP-1₍₇₋₃₇₎ content Arg³⁴GLP-1₍₉₋₃₇₎ content Samplefor 36% 13% application Impurity peak  2% 45% Main peak 50%  1%

[0149] Chromatograms of the sample for application and the eluate isshown in FIGS. 4 and 5, respectively. The analytical results show aselective separation of the truncated form and a high reduction of thetruncated form in the main peak containing the target GLP-1 moiety bycation exchange chromatography employing organic modifiers.

Example 6

[0150] Arg³⁴GLP-1₍₇₋₃₇₎ was expressed and captured by cation exchange asdescribed in Example 1. The pool containing Arg³⁴GLP-1₍₇₋₃₇₎ and thetruncated form, Arg³⁴GLP-1₍₉₋₃₇₎, as an impurity was adjusted to pH 3.1,and 10 ml of the resulting solution was applied to a 20 ml Source 30S(Amersham Pharmacia Biotech) column equilibrated with 100 ml 20 mmol/kgcitric acid, 45% (w/w) ethanol, pH 3.0. The column was washed with 20 mlequilibration solution, and elution was performed with a linear saltgradient from 0 to 250 mmol/kg KCl (20 mmol/kg citric acid, 45% (w/w)ethanol, pH 3.0) followed by 60 ml of isocratic elution at 250 mmol/kgKCl. A chromatogram is shown in FIG. 6. Distinct peaks and separationbetween the truncated form and the target GLP-1 moiety were obtained byaddition of ethanol to the chromatographic solutions.

Example 7

[0151] Arg³⁴GLP-1₍₇₋₃₇₎ was expressed and captured by cation exchange asdescribed in Example 1. The pool containing Arg³⁴GLP-1₍₇₋₃₇₎ and thetruncated form, Arg³⁴GLP-1₍₉₋₃₇₎, as an impurity was adjusted to pH 3.5,and 10 ml of the resulting solution was applied to a 20 ml Source 30S(Amersham Pharmacia Biotech) column equilibrated with 100 ml 20 mmol/kgcitric acid, 45% (w/w) ethanol, pH 3.5. The column was washed with 20 mlequilibration solution, and elution was performed with a linear saltgradient from 0 to 250 mmol/kg KCl (20 mmol/kg citric acid, 45% (w/w)ethanol, pH 3.5) followed by 40 ml of isocratic elution at 250 mmol/kgKCl. Distinct peaks and separation between the truncated form and thetarget GLP-1 moiety similar to Example 6 were obtained.

Example 8

[0152] Arg³⁴GLP-1₍₇₋₃₇₎ fermentation broth was purified by aconventional cation exchange chromatography capture step followed by aconventional RP-HPLC purification step. 6 volumes of water was added tothe pool containing Arg³⁴GLP-1₍₇₋₃₇₎ and the truncated form,Arg³⁴GLP-1₍₉₋₃₇₎, as an impurity, and the solution was adjusted to pH3.5. 170 ml of the resulting solution was applied to a 20 ml Source 30S(Amersham Pharmacia Biotech) column equilibrated with 100 ml 20 mmol/kgcitric acid, 37.5 mmol/kg KCl, 45% (w/w) ethanol, pH 3.5. The column waswashed with 20 ml equilibration solution, and elution was performed witha linear salt gradient from 37.5 to 162.5 mmol/kg KCl (20 mmol/kg citricacid, 45% (w/w) ethanol, pH 3.5) followed by 20 ml of isocratic elutionat 250 mmol/kg KCl (20 mmol/kg citric acid, 45% (w/w) ethanol, pH 3.5).Distinct peaks and separation between the truncated form and the targetGLP-1 moiety were obtained by addition of ethanol to the chromatographicsolutions.

Example 9

[0153] Arg³⁴GLP-1₍₇₋₃₇₎ was expressed and captured by cation exchange asdescribed in Example 1. 10 ml of the pool containing Arg³⁴GLP-1₍₇₋₃₇₎and various impurities was applied to a 20 ml DEAE HyperD 20 (BioSepraS. A.) column equilibrated with 100 ml 20 mM di-sodium hydrogenphosphate di-hydrate, pH 7.5. The column was washed with 20 mlequilibration solution, and elution was performed with a linear saltgradient from 0 to 250 mM NaCl (20 mM di-sodium hydrogen phosphatedi-hydrate, pH 7.5) followed by 40 ml of isocratic elution with 1 M NaCl(20 mM di-sodium hydrogen phosphate di-hydrate, pH 7.5). No distinctpeaks or separation were obtained as the target GLP-1 moiety was elutingduring all of the peak area.

Example 10

[0154] Arg³⁴GLP-1₍₇₋₃₇₎ was expressed and captured by cation exchange asdescribed in Example 1. The pool containing Arg³⁴GLP-1₍₇₋₃₇₎ and variousimpurities was diluted with three volumes of water, and 40 ml of theresulting solution was applied to a 20 ml Source 15Q (Amersham PharmaciaBiotech) column equilibrated with 100 ml 20 mM Tris-hydroxymethylamino-methane, pH 8.5. The column was washed with 20 ml equilibrationsolution, and elution was performed with a linear salt gradient from 0to 250 mM NaCl (20 mM Tris-hydroxymethyl amino-methane, pH 8.5) followedby 40 ml of isocratic elution with 250 mM NaCl. A chromatogram is shownin FIG. 7. No distinct peaks or separation were obtained as the targetGLP-1 moiety was eluting during all of the peak area.

Example 11

[0155] Arg³⁴GLP-1₍₇₋₃₇₎ was expressed and captured by cation exchange asdescribed in Example 1. The pool containing Arg³⁴GLP-1₍₇₋₃₇₎ and variousimpurities was diluted with three volumes of water, and 20 ml of theresulting solution was applied to a 20 ml Source 15Q (Amersham PharmaciaBiotech) column equilibrated with 100 ml 20 mmol/kg Tris-hydroxymethylamino-methane, 45% (w/w) ethanol, pH 8.5. The column was washed with 20ml equilibration solution, and elution was performed with a linear saltgradient from 0 to 250 mmol/kg NaCl (20 mmol/kg Tris-hydroxymethylamino-methane, 45% (w/w) ethanol, pH 8.5) followed by 40 ml of isocraticelution with 250 mmol/kg NaCl. A chromatogram is shown in FIG. 8.Distinct peaks and separation between various impurities and the targetGLP-1 moiety were obtained by addition of ethanol to the chromatographicsolutions. Insignificant differences in set up between this and Example10 are: different load and buffer concentration.

Example 12

[0156] Arg³⁴GLP-1₍₇₋₃₇₎ was expressed and captured by cation exchange asdescribed in Example 1. The pool containing Arg³⁴GLP-1₍₇₋₃₇₎ and variousimpurities was diluted with one volume of water and two volumes ofethanol, and 20 ml of the resulting solution was applied to a 20 mlSource 15Q (Amersham Pharmacia Biotech) column equilibrated with 100 ml20 mmol/kg Tris-hydroxymethyl amino-methane, 45% (w/w) ethanol, pH 8.5.The column was washed with 20 ml equilibration solution, and elution wasperformed with a linear salt gradient from 0 to 100 mmol/kg NaCl (20mmol/kg Tris-hydroxymethyl amino-methane, 45% (w/w) ethanol, pH 8.5)followed by 40 ml of isocratic elution with 100 mM NaCl. Distinct peaksand separation between various impurities and the target GLP-1 moietywere obtained by addition of ethanol to the chromatographic solutions.

Example 13

[0157] Arg³⁴GLP-1₍₇₋₃₇₎ was expressed as described in Example 1.Arg³⁴GLP-1₍₇₋₃₇₎ was isolated from the fermentation broth by aconventional reverse phase chromatography capture step, and subsequentlyprecipitated at the pI (isoelectric point) of Arg³⁴GLP-1₍₇₋₃₇₎. 10 g ofthe precipitate containing Arg³⁴GLP-1₍₇₋₃₇₎ and the truncated form,Arg³⁴GLP-1₍₉₋₃₇₎, as one of several impurities was suspended in 500 mlwater and dissolved by pH adjustment to 8.3 to a Arg³⁴GLP-1₍₇₋₃₇₎concentration of approximately 1.6 mg/ml. 5 ml of the resulting solutionwas adjusted to pH 3.5 and applied to a 20 ml Source 30S (AmershamPharmacia Biotech) column equilibrated with 60 ml 0.42% w/w citric acid,pH 3.5. The truncated form was not eluted/washed off by a lineargradient from 0 to 2 M NaCl (0.42% w/w citric acid, pH 3.5). The targetpeptide, Arg³⁴GLP-1₍₇₋₃₇₎, and the impurity, Arg³⁴GLP-1₍₉₋₃₇₎, wereeluted in a single peak by 40 ml of the regeneration solvent 4% w/wNaOH. A chromatogram is shown in FIG. 9. No removal of the truncatedimpurity was achieved by the wash step at low pH with a conventionalhigh salt solution without an organic modifier.

Example 14

[0158] Arg³⁴GLP-1₍₇₋₃₇₎ was isolated from the fermentation broth byconventional reverse phase chromatography and precipitated as describedin Example 13. 10 g of the precipitate containing Arg³⁴GLP-1₍₇₋₃₇₎ andthe truncated form, Arg³⁴GLP-1₍₉₋₃₇₎, as one of several impurities wassuspended in 500 ml water and dissolved by pH adjustment to 8.3 to aArg³⁴GLP-1₍₇₋₃₇₎ concentration of approximately 1.6 mg/ml. 5 ml of theresulting solution was adjusted to pH 3.5 and applied to a 20 ml Source30S (Amersham Pharmacia Biotech) column equilibrated with 60 ml 0.42%w/w citric acid, 34% w/w ethanol, pH 3.5. Elution was performed with alinear salt gradient from 0 to 2.23% w/w KCl (0.42% w/w citric acid, 34%w/w ethanol, pH 3.5). Distinct peaks and separation between thetruncated form and the target GLP-1 moiety were obtained similar toExample 6.

Example 15

[0159] Arg³⁴GLP-1₍₇₋₃₇₎ was isolated from the fermentation broth byconventional reverse phase chromatography and precipitated as describedin Example 13. 10 g of the precipitate containing Arg³⁴GLP-1₍₇₋₃₇₎ andthe truncated form, Arg³⁴GLP-1₍₉₋₃₇₎, as one of several impurities wassuspended in 500 ml water and dissolved by pH adjustment to 8.3 to aArg³⁴GLP-1₍₇₋₃₇₎ concentration of approximately 1.6 mg/ml. 5 ml of theresulting solution was adjusted to pH 3.5 and applied to a 20 ml Source30S (Amersham Pharmacia Biotech) column equilibrated with 60 ml 0.42%w/w citric acid, 29% w/w ethanol, pH 3.5. Elution was performed with alinear salt gradient from 0 to 2.23% w/w KCl (0.42% w/w citric acid, 29%w/w ethanol, pH 3.5). Separation between the truncated form and thetarget GLP-1 moiety were obtained.

Example 16

[0160] Arg³⁴GLP-1₍₇₋₃₇₎ was isolated from the fermentation broth byconventional reverse phase chromatography and precipitated as describedin Example 13. 10 g of the precipitate containing Arg³⁴GLP-1₍₇₋₃₇₎ andthe truncated form, Arg³⁴GLP-1₍₉₋₃₇₎, as one of several impurities wassuspended in 500 ml water and dissolved by pH adjustment to 8.3 to aArg³⁴GLP-1₍₇₋₃₇₎ concentration of approximately 1.6 mg/ml. 5 ml of theresulting solution was adjusted to pH 3.5 and applied to a 20 ml Source30S (Amersham Pharmacia Biotech) column equilibrated with 60 ml 0.42%w/w citric acid, 51% w/w ethanol, pH 3.5. Elution was performed with alinear salt gradient from 0 to 2.23% w/w KCl (0.42% w/w citric acid, 51%w/w ethanol, pH 3.5). Distinct peaks and separation between thetruncated form and the target GLP-1 moiety were obtained similar toExample 6.

Example 17

[0161] Arg³⁴GLP-1₍₇₋₃₇₎ was isolated from the fermentation broth byconventional reverse phase chromatography and precipitated as describedin Example 13. 10 g of the precipitate containing Arg³⁴GLP-1₍₇₋₃₇₎ andthe truncated form, Arg³⁴GLP-1₍₉₋₃₇₎, as one of several impurities wassuspended in 500 ml water and dissolved by pH adjustment to 8.3 to aArg³⁴GLP-1₍₇₋₃₇₎ concentration of approximately 1.6 mg/ml. 5 ml of theresulting solution was adjusted to pH 3.5 and applied to a 20 ml Source30S (Amersham Pharmacia Biotech) column equilibrated with 60 ml 0.42%w/w citric acid, 71% w/w ethanol, pH 3.5. Elution was performed with alinear salt gradient from 0 to 1.12% w/w KCl (0.42% w/w citric acid, 71%w/w ethanol, pH 3.5). A chromatogram is shown in FIG. 10. Distinct peaksand separation between the truncated form and the target GLP-1 moietywere obtained similar to Example 6.

Example 18

[0162] Arg³⁴GLP-1₍₇₋₃₇₎ was isolated from the fermentation broth byconventional reverse phase chromatography and precipitated as describedin Example 13. 10 g of the precipitate containing Arg³⁴GLP-1₍₇₋₃₇₎ andthe truncated form, Arg³⁴GLP-1₍₉₋₃₇₎, as one of several impurities wassuspended in 500 ml water and dissolved by pH adjustment to 8.3 to aArg³⁴GLP-1₍₇₋₃₇₎ concentration of approximately 1.6 mg/ml. 5 ml of theresulting solution was adjusted to pH 3.5 and applied to a 20 ml Source30S (Amersham Pharmacia Biotech) column equilibrated with 60 ml 0.42%w/w citric acid, 40% w/w 2-propanol, pH 3.5. Elution was performed witha linear salt gradient from 0 to 2.23% w/w KCl (0.42% w/w citric acid,40% w/w 2-propanol, pH 3.5). A chromatogram is shown in FIG. 11.Distinct peaks and separation between the truncated form and the targetGLP-1 moiety were obtained.

Example 19

[0163] Arg³⁴GLP-1₍₇₋₃₇₎ was isolated from the fermentation broth byconventional reverse phase chromatography and precipitated as describedin Example 13. 10 g of the precipitate containing Arg³⁴GLP-1₍₇₋₃₇₎ andthe truncated form, Arg³⁴GLP-1₍₉₋₃₇₎, as one of several impurities wassuspended in 500 ml water and dissolved by pH adjustment to 8.3 to aArg³⁴GLP-1₍₇₋₃₇₎ concentration of approximately 1.6 mg/ml. 5 ml of theresulting solution was adjusted to pH 3.5 and applied to a 8 ml Poros 50HS (PE Biosystems) column equilibrated with 24 ml 0.42% w/w citric acid,51% w/w ethanol, pH 3.5. Elution was performed with a linear saltgradient from 0 to 2.23% w/w KCl (0.42% w/w citric acid, 51% w/wethanol, pH 3.5). A chromatogram is shown in FIG. 12. Distinct peaks andseparation between the truncated form and the target GLP-1 moiety wereobtained.

Example 20

[0164] Arg³⁴GLP-1₍₇₋₃₇₎ was isolated from the fermentation broth byconventional reverse phase chromatography and precipitated as describedin Example 13. 10 g of the precipitate containing Arg³⁴GLP-1₍₇₋₃₇₎ andthe truncated form, Arg³⁴GLP-1₍₉₋₃₇₎, as one of several impurities wassuspended in 500 ml water and dissolved by pH adjustment to 8.3 to aArg³⁴GLP-1₍₇₋₃₇₎ concentration of approximately 1.6 mg/ml. 5 ml of theresulting solution was adjusted to pH 3.5 and applied to a 8 ml Poros 50HS (PE Biosystems) column equilibrated with 24 ml 0.42% w/w citric acid,40% w/w 2-propanol, pH 3.5. Elution was performed with a linear saltgradient from 0 to 2.23% w/w KCl (0.42% w/w citric acid, 40% w/w2-propanol, pH 3.5). Distinct peaks and separation between the truncatedform and the target GLP-1 moiety were obtained.

Example 21

[0165] Arg³⁴GLP-1₍₇₋₃₇₎ was isolated from the fermentation broth byconventional reverse phase chromatography and precipitated as describedin Example 13. 10 of the precipitate containing Arg³⁴GLP-1₍₇₋₃₇₎ and thetruncated form, Arg³⁴GLP-1₍₉₋₃₇₎, as one of several impurities wassuspended in 500 ml water and dissolved by pH adjustment to 8.3 to aArg³⁴GLP-1₍₇₋₃₇₎ concentration of approximately 1.6 mg/ml. 5 ml of theresulting solution was adjusted to pH 3.5 and applied to a 20 ml Source30S (Amersham Pharmacia Biotech) column equilibrated with 60 ml 0.42%w/w citric acid, 40% w/w 2-methyl-2,4-pentanediol, pH 3.5. Elution wasperformed with a linear salt gradient from 0 to 2.23% w/w KCl (0.42% w/wcitric acid, 40% w/w 2-methyl-2,4-pentanediol, pH 3.5). A chromatogramis shown in FIG. 13. Distinct peaks and separation between the truncatedform and the target GLP-1 moiety were obtained.

Example 22

[0166] Arg³⁴GLP-1₍₇₋₃₇₎ was isolated from the fermentation broth byconventional reverse phase chromatography and precipitated as describedin Example 13. The precipitate was dissolved in water and purified bycation exchange chromatography using an organic modifier followed by aconventional reverse phase chromatography step in ethanol. PurifiedArg³⁴GLP-1₍₇₋₃₇₎ from the reverse phase eluate was precipitated at thepI of Arg³⁴GLP-1₍₇₋₃₇₎. Arg³⁴GLP-1₍₇₋₃₇₎ was acylated as described in WO9808871. The resulting solution containing mono-acylatedArg³⁴GLP-1₍₇₋₃₇₎ in a concentration of 2 mg/ml, and Arg³⁴GLP-1₍₇₋₃₇₎ anddi-acylated Arg³⁴GLP-1₍₇₋₃₇₎ as impurities, was diluted with 3 volumesof water. 5 ml of the diluted solution with pH 6.9 was applied to a 20ml Source 30S (Amersham Pharmacia Biotech) column equilibrated with 60ml 0.42% w/w citric acid, 64.5% w/w ethanol, pH 3.5. Elution wasperformed with a linear salt gradient from 0 to 1.30% w/w KCl (0.42% w/wcitric acid, 64.5% w/w ethanol, pH 3.5). A chromatogram is shown in FIG.14. The GLP-1 components eluted as distinct peaks and separation betweenthe three GLP-1 moieties was obtained. RP-HPLC analysis foridentification/verification of collected peaks was carried out on adi-methyl-butyl-dimethyl-silyl substituted 100 Å silica (Fuji-Davison)4.0×250 mm column with 5 μm particles. Buffer A consisted of 0.15 M(NH₄)₂SO₄ in 7.8% (w/w) acetonitrile, pH 2.5, and buffer B contained63.4% (w/w) acetonitrile. The gradient program at a flow rate of 1ml/min was as follows: linear gradient from 35-57.5% B in 10 min, lineargradient from 57.5-67.5% B in 22 min, linear gradient from 67.5-90% B in3 min, isocratic at 90% B in 5 min, linear gradient from 90-35% B in 2min, and isocratic at 35% B in 5 min. The chromatographic temperaturewas kept at 60° C. and UV detection was performed at 214 nm. Theanalytical chromatograms verified the separation of the three GLP-1moieties.

Example 23

[0167] Arg³⁴GLP-1₍₇₋₃₇₎ was isolated from the fermentation broth byconventional reverse phase chromatography and precipitated as describedin Example 13. The precipitate was dissolved in water and purified bycation exchange chromatography using an organic modifier followed by aconventional reverse phase chromatography step in ethanol. PurifiedArg³⁴GLP-1₍₇₋₃₇₎ from the reverse phase eluate was precipitated at thepI of Arg³⁴GLP-1₍₇₋₃₇₎. Arg³⁴GLP-1₍₇₋₃₇₎ was acylated as described in WO9808871.

[0168] The resulting solution containing mono-acylated Arg³⁴GLP-1₍₇₋₃₇₎in a concentration of 2 mg/ml, and Arg³⁴GLP-1₍₇₋₃₇₎ and di-acylatedArg³⁴GLP-1₍₇₋₃₇₎ as impurities, was diluted with 3 volumes of water. 5ml of the diluted solution with pH 6.9 was applied to a 20 ml Source 30S(Amersham Pharmacia Biotech) column equilibrated with 60 ml 0.42% w/wcitric acid, 40% w/w 2-propanol, pH 3.5. Elution was performed with alinear salt gradient from 0 to 2.23% w/w KCl (0.42% w/w citric acid, 40%w/w 2-propanol, pH 3.5). Separation between the three GLP-1 moieties wasobtained. The RP-HPLC analysis method of Example 22 was used foridentification/verification of collected peaks.

1. A cation exchange chromatography process for purifying a peptide orGLP-1 peptide from a mixture comprising said peptide or GLP-1 peptideand related or unrelated impurities, comprising the step of: separatingsaid peptide or GLP-1 peptide and said related or unrelated impuritiesof said mixture by elution in a solution comprising an organic modifier,water, optionally a salt component, and optionally a buffer, with alinear or step gradient or isocratically in salt component and/or with alinear or step pH-gradient or at a constant pH-value, wherein thepH-gradient or pH-value should be in the range where said peptide orGLP-1 peptide has a positive local or overall net charge different fromthe local or overall positive net charge of said related or unrelatedimpurities so as to remove said related or unrelated impurities.
 2. Acation exchange chromatography process for purifying a peptide or GLP-1peptide from a mixture comprising said peptide or GLP-1 peptide andrelated impurities, comprising the step of: separating said peptide orGLP-1 peptide and said related impurities of said mixture by elution ina solution consisting essentially of an organic modifier, water,optionally a salt component, and optionally a buffer, with a linear orstep gradient or isocratically in salt component and/or with a linear orstep pH-gradient or at a constant pH-value, wherein the pH-gradient orpH-value should be in the range where said peptide or GLP-1 peptide hasa positive local or overall net charge different from the local oroverall positive net charge of said related impurities so as to removesaid related impurities.
 3. An anion exchange chromatography process forpurifying a peptide or GLP-1 peptide from a mixture comprising saidpeptide or GLP-1 peptide and related or unrelated impurities, comprisingthe step of: separating said peptide or GLP-1 peptide and said relatedor unrelated impurities of said mixture by elution in a solutioncomprising an organic modifier, water, optionally a salt component, andoptionally a buffer, with a linear or step gradient or isocratically insalt component and/or with a linear or step pH-gradient or at a constantpH-value, wherein the pH-gradient or pH-value should be in the rangewhere said peptide or GLP-1 peptide has a negative local or overall netcharge different from the local or overall negative net charge of saidrelated or unrelated impurities so as to remove said related orunrelated impurities.
 4. An anion exchange chromatography process forpurifying a peptide or GLP-1 peptide from a mixture comprising saidpeptide or GLP-1 peptide and related impurities, comprising the step of:separating said peptide or GLP-1 peptide and said related impurities ofsaid mixture by elution in a solution consisting essentially of anorganic modifier, water, optionally a salt component, and optionally abuffer, with a linear or step gradient or isocratically in saltcomponent and/or with a linear or step pH-gradient or at a constantpH-value, wherein the pH-gradient or pH-value should be in the rangewhere said peptide or GLP-1 peptide has a negative local or overall netcharge different from the local or overall negative net charge of saidrelated impurities so as to remove said related impurities.
 5. Anindustrial method for producing a pure peptide or GLP-1 peptide, themethod including a cation exchange chromatography process for purifyinga peptide from a mixture comprising said peptide or GLP-1 peptide andrelated impurities, comprising the steps of: separating said peptide orGLP-1 peptide and said related impurities of said mixture by elution ina solution comprising an organic modifier, water, optionally a saltcomponent, and optionally a buffer, with a linear or step gradient orisocratically in salt component and/or with a linear or step pH-gradientor at a constant pH-value, wherein the pH-gradient or pH-value should bein the range where said peptide or GLP-1 peptide has a positive local oroverall net charge different from the local or overall positive netcharge of said related impurities so as to remove said relatedimpurities.
 6. An industrial method for producing a pure peptide orGLP-1 peptide, the method including an anion exchange chromatographyprocess for purifying a peptide from a mixture comprising said peptideor GLP-1 peptide and related impurities, comprising the steps of:separating said peptide or GLP-1 peptide and said related impurities ofsaid mixture by elution in a solution comprising an organic modifier,water, optionally a salt component, and optionally a buffer, with alinear or step gradient or isocratically in salt component and/or with alinear or step pH-gradient or at a constant pH-value, wherein thepH-gradient or pH-value should be in the range where said peptide orGLP-1 peptide has a negative local or overall net charge different fromthe local or overall negative net charge of said related impurities soas to remove said related impurities.
 7. A method for isolating apeptide or GLP-1 peptide, the method including purification of a peptideor GLP-1 peptide from a mixture comprising said peptide or GLP-1 peptideand related impurities via a cation exchange chromatography process, thecation exchange chromatography process comprising the steps of:separating said peptide or GLP-1 peptide and said related impurities ofsaid mixture by elution in a solution comprising an organic modifier,water, optionally a salt component, and optionally a buffer, with alinear or step gradient or isocratically in salt component and/or with alinear or step pH-gradient or at a constant pH-value, wherein thepH-gradient or pH-value should be in the range where said peptide orGLP-1 peptide has a positive local or overall net charge different fromthe local or overall positive net charge of said related impurities soas to remove said related impurities; and subsequently, if necessary,subjecting to analytical tests and/or further purification, andisolating said peptide or GLP-1 peptide in a conventional manner.
 8. Amethod for isolating a peptide or GLP-1 peptide, the method includingpurification of a peptide or GLP-1 peptide from a mixture comprisingsaid peptide or GLP-1 peptide and related impurities via an anionexchange chromatography process, the anion exchange chromatographyprocess comprising the steps of: separating said peptide or GLP-1peptide and said related impurities of said mixture by elution in asolution comprising an organic modifier, water, optionally a saltcomponent, and optionally a buffer, with a linear or step gradient orisocratically in salt component and/or with a linear or step pH-gradientor at a constant pH-value, wherein the pH-gradient or pH-value should bein the range where said peptide or GLP-1 peptide has a negative local oroverall net charge different from the local or overall negative netcharge of said related impurities so as to remove said relatedimpurities; and subsequently, if necessary, subjecting to analyticaltests and/or further purification, and isolating said peptide or GLP-1peptide in a conventional manner.
 9. The process or method according toany one of claims 1-8 wherein said GLP-1 peptide to be purified isselected from GLP-1 (7-37), GLP-1 (7-36) amide as well as analogues andderivatives thereof, such as Arg³⁴GLP-1(7-37); Arg²⁶GLP-1(7-37);Arg³⁴Lys²⁶(N^(ε)-(γ-Glu-(N^(α)-tetradecanoyl)))GLP-1 (7-37);Arg³⁴Lys²⁶(N^(ε)-(γ-Glu-(N^(α)-hexadecanoyl)))GLP-1 (7-37);Arg²⁶Lys³⁴(N^(ε)-(γ-Glu-(N^(α)-tetradecanoyl)))GLP-1 (7-37);Arg²⁶Lys³⁴(N^(ε)-(γ-Glu-(N^(α)-hexadecanoyl)))GLP-1 (7-37);Val⁸GLP-1(7-37); Thr⁸GLP-1(7-37); Met⁸GLP-1(7-37); Gly⁸GLP-1(7-37);Val⁸GLP-1(7-36) amide; Thr⁸GLP-1(7-36) amide; Met⁸GLP-1(7-36) amide;Gly⁸GLP-1(7-36) amide.
 10. The process or method according to any one ofclaims 1-8 wherein said peptide to be purified is selected fromFactorVII, FactorVIIa, FactorVIIai, and FFR-FactorVIIa.
 11. The processor method according to any one of claims 1-8 wherein said peptide to bepurified is selected from a GLP-2 peptide as well as analogues andderivatives thereof.
 12. The process or method according to any one ofclaims 1-11 wherein the ratio of organic modifier to water on a weightpercent basis is from 1:99 to 99:1.
 13. The process or method accordingto any one of claims 1-12 wherein said organic modifier is selected fromC₁₋₆-alkanol, C₁₋₆-alkenol or C₁₋₆-alkynol, urea, guanidine, orC₁₋₆-alkanoic acid, C₂₋₆-glycol, or C₃₋₇-polyalcohol including sugars.